Activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mammalian target of rapamycin (mTOR) kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners towards regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor and Rictor mRNA was noted with advanced stages of CRC suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastasis compared to normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact as well as decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating EMT, motility and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.

Purpose: The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism, and cytoskeleton. Because ∼60% of sporadic colorectal cancers (CRC) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs. Experimental Design: HCT116, KM20, Caco-2, and SW480 human CRC cells were used to determine the effects of pharmacologic (using rapamycin) or genetic (using RNAi) blockade of mTOR signaling on cell proliferation, apoptosis, cell cycle progression, and subcutaneous growth in vivo. Results: We show that the mTOR complex proteins mTOR, Raptor, and Rictor are overexpressed in CRC. Treatment with rapamycin significantly decreased proliferation of certain CRC cell lines (rapamycin sensitive), whereas other cell lines were resistant to its effects (rapamycin resistant). Transient siRNA-mediated knockdown of the mTORC2 protein, Rictor, significantly decreased proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells. Stable shRNA-mediated knockdown of both mTORC1 and mTORC2 decreased proliferation, increased apoptosis, and attenuated cell cycle progression in rapamycin-sensitive CRCs. Moreover, stable knockdown of both mTORC1 and mTORC2 decreased proliferation and attenuated cell cycle progression, whereas only mTORC2 knockdown increased apoptosis in rapamycin-resistant CRCs. Finally, knockdown of both mTORC1 and mTORC2 inhibited growth of rapamycinsensitive and rapamycin-resistant CRCs in vivo when implanted as tumor xenografts. Conclusions: Targeted inhibition of the mTORC2 protein, Rictor, leads to growth inhibition and induces apoptosis in both rapamycin-sensitive and rapamycin-resistant CRCs, suggesting that selective targeting of mTORC2 may represent a novel therapeutic strategy for treatment of CRC. (Clin Cancer Res 2009;15(23):7207-16)