The MEK/ERK and PI3K/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eIF4E-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-BP1 for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mTORC1-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally-regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC.

Mobilization of intracellular Ca2ϩ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca 2ϩ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca 2ϩ

Sphingosine 1-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Contradictory reports propose that S1P acts as either an intracellular second messenger or an extracellular ligand for cell-surface receptors. Hence, the precise signaling mechanisms mediating the diverse cellular effects of S1P remain to be determined. Here, we investigate whether S1P stimulation of cell proliferation, survival, and related signaling events can be mediated by the recently cloned Edg family members of G protein-coupled receptors. We observed that S1P treatment significantly increased proliferation of HTC4 hepatoma cells stably transfected with human S1P receptor Edg3 or Edg5, which was attributable to stimulation of cell growth and inhibition of apoptosis caused by serum starvation. Edg3 and Edg5 transduced S1P-evoked signaling events relevant to cell proliferation and survival, including activation of the ERK/MAP kinases, and immediate-early induction of c-Jun and c-Fos. Trancriptional activation of reporter genes for the c-fos promoter and the serum response element by Edg3 and Edg5 transfected in Jurkat cells was inhibited by pertussis toxin and C3 exoenzyme, implicating G i/o -and Rho-dependent pathways. Our data also indicated that Edg3 and Edg5 mediated the serum response element activation through transcriptional factors Elk-1 and serum response factor. Thus, specific G protein-coupled receptors Edg3 and Edg5 account for, at least in part, S1P-induced cell proliferation, survival, and related signaling events.Several types of sphingolipids, such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P), 1 have attracted increasing attention as mediators of important cellular functions (1-3). S1P, in particular, is implicated in cell proliferation (4 -6), suppression of apoptosis (7,8), modulation of cell motility, tumor invasiveness (9, 10), platelet activation (11), and neurite retraction (12). Cellular signaling by S1P evokes activation of the mitogen-activated protein kinases (MAP kinases) (9, 13), inward rectifying K ϩ channels (I k(Ach) ) (14, 15), and intracellular Ca 2ϩ mobilization (4, 14, 16 -20). Despite extensive observations in various cell types, the precise signaling mechanisms by which S1P exerts its cellular effects remain undetermined. The major uncertainty is whether S1P acts intracellularly as a second messenger, or extracellularly as a receptor ligand, or both. The second messenger hypothesis is based mainly on the following observations: first, S1P stimulated Ca 2ϩ release directly from isolated endoplasmic reticulum preparations through an inositol trisphosphate-independent mechanism (17, 18); second, inhibition of S1P production by sphingosine kinase inhibitors, e.g. N,N-dimethylsphingosine or dihydrosphingosine, blocked cell proliferation induced by platelet-derived growth factor or serum, and Ca 2ϩ mobilization induced by IgE or muscarinic receptor stimulation (21,22). These observations, together with the finding that the intracellular level of S1P was eleva...

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