Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), inhibits tumor cell motility. However, the underlying mechanism is poorly understood. Here, we show that rapamycin inhibited type I insulin-like growth factor (IGF-I)-stimulated motility of a panel of cell lines. Expression of a rapamycin-resistant mutant of mTOR (mTORrr) prevented rapamycin inhibition of cell motility. However, cells expressing a kinase-dead mTORrr remained sensitive to rapamycin. Downregulation of raptor or rictor by RNA interference (RNAi) decreased cell motility. However, only downregulation of raptor mimicked the effect of rapamycin, inhibiting phosphorylation of S6 kinase 1 (S6K1) and 4E-BP1. Cells infected with an adenovirus expressing constitutively active and rapamycin-resistant mutant of p70 S6K1, but not with an adenovirus expressing wild-type S6K1, or a control virus, conferred to resistance to rapamycin. Further, IGF-I failed to stimulate motility of the cells, in which S6K1 was downregulated by RNAi. Moreover, downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) by RNAi-attenuated rapamycin inhibition of cell motility. In contrast, expression of constitutively active 4E-BP1 dramatically inhibited IGF-I-stimulated cell motility. The results indicate that both S6K1 and 4E-BP1 pathways, regulated by TORC1, are required for cell motility. Rapamycin inhibits IGF-I-stimulated cell motility, through suppression of both S6K1 and 4E-BP1/eIF4E-signaling pathways, as a consequence of inhibition of mTOR kinase activity.

The alteration of p21-activated kinase 4 (PAK4) and transforming growth factor-beta (TGF-β) signaling effector Smad2/3 was detected in several types of tumors, which acts as oncogenic factor and tumor suppressor, but the relationship between these events has not been explored. Here, we demonstrate that PAK4 interacts with and modulates phosphorylation of Smad2/3 via both kinase-dependent and kinase-independent mechanisms, which attenuate Smad2/3 axis transactivation and TGF-β-mediated growth inhibition in gastric cancer cells. First, PAK4 interaction with Smad2/3, which is independent of PAK4 kinase activity, blocks TGF-β1-induced phosphorylation of Smad2 Ser465/467 or Smad3 Ser423/425 and the consequent activation. In addition, PAK4 phosphorylates Smad2 on Ser465, leading to the degradation of Smad2 through ubiquitin-proteasome-dependent pathway under hepatocyte growth factor (HGF) stimulation. Interestingly, PAK4 expression correlates negatively with phospho-Ser465/467 Smad2 but positively with phospho-Ser465 Smad2 in gastric cancer tissues. Furthermore, the expressions of HGF, phospho-Ser474 PAK4 and phospho-Ser465 Smad2 are markedly increased in gastric cancer tissues, and the expression of Smad2 is decreased in gastric cancer tissues. Our results document an oncogenic role of PAK4 in repression of Smad2/3 transactivation that involved in tumorigenesis, and suggest PAK4 as a potential therapeutic target for gastric cancer.