Rapamycin inhibition of cell motility is associated with its suppression of synthesis of GTPases Free

2562

Rapamycin and its derivatives CCI-779, RAD001 and AP23573, inhibitors of the mammalian target of rapamycin (mTOR), are in clinical trials as novel anticancer agents. mTOR inhibitors not only suppress tumor cell proliferation and growth, but also inhibit tumor cell motility. Our recent findings have demonstrated that rapamycin inhibits type I insulin-like growth factor (IGF-I)-stimulated cell motility by inhibiting phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), two downstream effector molecules of mTOR-mLST8-raptor (mTORC1). Here we show that rapamycin inhibits the activities of GTPases (Rac1, cdc42 and RhoA), crucial regulatory proteins for cell migration. Rapamycin altered neither mRNA levels, nor turn-over of GTPases, but inhibited protein synthesis of these GTPases. Furthermore, we found that downregulation of raptor, rictor or mTOR by RNA interference mimicked effect of rapamycin, also reducing cellular protein levels of GTPases, suggesting both mTORC1 and mTOR-mLST8-rictor (mTORC2) are involved in the regulation of GTPases expression. Next, we further studied how the two mTOR complexes regulates the translation of the GTPases. Expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of expression of GTPases, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) suppressed expression of GTPases. mTOR-mLST8-rictor complex (mTORC2) regulates Akt activity. However, expression of constitutively active Akt did not influence rapamycin inhibition of GTPases. The data indicate that mTORC1 controls GTPases synthesis through S6K1 and 4E-BP1/eIF4E pathways, whereas mTORC2 controls GTPases synthesis through Akt-independent mechanism. Rapamycin inhibits cell motility possibly in part by downregulation of GTPases through mTORC1- and mTORC2-mediated signaling pathways. Supported by NIH/NCI CA115414 (SH).