ABSTRACT
Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors.
Subject(s)
Carrier Proteins/pharmacology , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/antagonists & inhibitors , Cell Proliferation/drug effects , Cell Survival/drug effects , Everolimus , Humans , Imidazoles/pharmacology , Immunoblotting , Immunosuppressive Agents/pharmacology , Insulin Receptor Substrate Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Neoplasms/drug therapy , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Kinases/chemistry , Protein Kinases/genetics , Proteins , Quinolines/pharmacology , RNA, Small Interfering/pharmacology , Rapamycin-Insensitive Companion of mTOR Protein , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Tumor Cells, CulturedABSTRACT
The bioenergetic response of B lymphocytes is subject to rapid changes following antigen encounter in order to provide ATP and anabolic precursors necessary to support growth. However, the pathways involved in glucose acquisition and metabolism are unknown. We find that B lymphocytes rapidly increase glucose uptake and glycolysis following B-cell antigen receptor (BCR) crosslinking. Inhibition of glycolysis blocks BCR-mediated growth. Prior to S-phase entry, glucose metabolism shifts from primarily glycolytic to include the pentose phosphate pathway. BCR-induced glucose utilization is dependent upon phosphatidylinositol 3-kinase (PI-3K) activity as evidenced by inhibition of glucose uptake and glycolysis with LY294002 treatment of normal B cells and impaired glucose utilization in B cells deficient in the PI-3K regulatory subunit p85alpha. Activation of Akt is sufficient to increase glucose utilization in B cells. We find that glucose utilization is inhibited by coengagement of the BCR and FcgammaRIIB, suggesting that limiting glucose metabolism may represent an important mechanism underlying FcgammaRIIB-mediated growth arrest. Taken together, these findings demonstrate that both growth-promoting BCR signaling and growth-inhibitory FcgammaRIIB signaling modulate glucose energy metabolism. Manipulation of these pathways may prove to be useful in the treatment of lymphoproliferative disorders, wherein clonal expansion of B lymphocytes plays a role.
