More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations, and are often refractory to approved targeted therapies. We report that cultured CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C depleting glutathione. Thus, ROS accumulates and inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibiting GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. In vivo studies indicate that high-dose vitamin C can impair tumor growth in Apc/KrasG12D mutant mouse intestinal cancers. While it is unclear whether human tumors will respond similarly, our results provide a mechanistic rationale for exploring the therapeutic use of vitamin C to treat CRCs with KRAS or BRAF mutations.

Rationale: Autophagy, a bulk degradation process of cytosolic proteins and organelles, is protective during nutrient starvation in cardiomyocytes (CMs). However, the underlying signaling mechanism mediating autophagy is not well understood. Objective:We investigated the role of FoxOs and its posttranslational modification in mediating starvationinduced autophagy. Methods and Results:Glucose deprivation (GD) increased autophagic flux in cultured CMs, as evidenced by increased mRFP-GFP-LC3 puncta and decreases in p62, which was accompanied by upregulation of Sirt1 and FoxO1. Overexpression of either Sirt1 or FoxO1 was sufficient for inducing autophagic flux, whereas both Sirt1 and FoxO1 were required for GD-induced autophagy. GD increased deacetylation of FoxO1, and Sirt1 was required for GD-induced deacetylation of FoxO1. Overexpression of FoxO1(3A/LXXAA), which cannot interact with Sirt1, or p300, a histone acetylase, increased acetylation of FoxO1 and inhibited GD-induced autophagy. FoxO1 increased expression of Rab7, a small GTP-binding protein that mediates late autophagosome-lysosome fusion, which was both necessary and sufficient for mediating FoxO1-induced increases in autophagic flux. Although cardiac function was maintained in control mice after 48 hours of food starvation, it was significantly deteriorated in mice with cardiac-specific overexpression of FoxO1(3A/LXXAA), those with cardiac-specific homozygous deletion of FoxO1 (c-FoxO1 Key Words: autophagy Ⅲ starvation Ⅲ FoxO Ⅲ Sirt1 Ⅲ Rab7 Ⅲ deacetylation M acroautophagy (termed hereafter as autophagy) is a dynamic process of intracellular bulk degradation in which cytosolic proteins and organelles are sequestered into double-membrane vesicles called autophagosomes to be fused with lysosomes for degradation. 1 In the heart, autophagy maintains protein quality control, adapts to nutrient and oxygen deprivation during myocardial ischemia, and mediates cell death during reperfusion injury. 2,3 Autophagy during nutrient deprivation is an adaptive mechanism that allows the cells to survive by degrading the intracellular protein and lipid cargo and recycling the amino and fatty acids to generate ATP. 2 The nutrient status has a profound effect on cardiac contractility, and activation of autophagy during starvation is protective for the heart. The intracellular signaling mechanism by which nutrient starvation activates autophagy in cardiomyocytes (CMs) is not well understood, however.The forkhead box, class O (FoxO) family of transcription factors are present as 4 distinct isoforms (FoxO1, FoxO3, FoxO4, and FoxO6) in mammals. FoxO proteins play an important role in several intracellular functions, such as metabolism, stress resistance, longevity, tumor suppression, and cell size regulation. 4 The key to the myriad functions of FoxO proteins lies in the complex posttranslational modifications they undergo. They are phosphorylated in response to insulin and growth factors, dephosphorylated by protein phosphatases, ubiquitinated in response to oxidative stre...

FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for sphingosine 1-phosphate (S1P) receptors. The effects of FTY720 on vascular cells, a major target of S1P action, have not been addressed. We now report the metabolic activation of FTY720 by sphingosine kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by sphingosine kinase activity and formation of FTY720-P. Sphingosine kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for G i -coupled S1P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth.

[Keywords: sphingosine 1-phosphate; cadherin; angiogenesis; vascular stabilization; endothelial cells; pericytes] Supplemental material is available at http://www.genesdev.org.

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/؉ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/؉ Sphk ؊/؊ mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/؉ S1p2r ؊/؊ , Apc Min/؉ S1p3r ؊/؊ , and Apc Min/؉ S1p1r ؉/؊ bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/؉ Sphk1 ؊/؊ mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G 1 /S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/؉ Sphk1 ؊/؊ mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G 1 /S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.The concept that sphingolipid metabolism is an important source of signaling lipids has gained considerable acceptance. For example, sphingomyelin, an abundant membrane phospholipid, is metabolized into lipid mediators such as sphingosine, ceramide, ceramide 1-phosphate, and sphingosine 1-phosphate (S1P) (9). S1P, synthesized by the action of sphingosine kinase (Sphk), is secreted by cells and functions as an extracellular mediator by activating a family of G-protein-coupled receptors termed S1PR1-5 (13, 19, 30). The generality of S1P signaling in vertebrates is underscored by recent findings that S1PRs are needed for a multitude of physiological processes, including heart and vascular development, angiogenesis, and immune cell trafficking (12). However, the physiological and pathological significance of intracellular sphingolipid metabolism in vertebrates is virtually unknown. However, a recent study showed that the inhibition of S1P lyase resulted in alterations in tissue S1P levels, which influenced T-cell trafficking and immune responses (35). The importance of sphingolipid metabolism is underscored by its impact on cell death, stress responses, metabolism, and animal development in unicellular (Saccharomyces cerevisiae and Dictyostelium discoidium) and multicellular (Caenorhabditis elegans and Drosophila melanogaster) eukaryotes (4,5,10,21,26,28).Mammals express two functiona...

Sphingosine 1-phosphate (S1P) is a lysophospholipid that exerts a variety of responses in cells such as proliferation, migration, and survival. These effects are mediated by G protein-coupled receptors on the cell surface (S1P 1-5 ), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of S1P action in human glioblastoma cells are not well defined. S1P receptors (1-5) and S1P-metabolizing enzymes were expressed in three human glioblastoma cell lines. S1P had a profound and differential effect on glioblastoma cell migration. U87 cells treated with S1P showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited. S1P-mediated inhibition correlated with S1P 2 receptor expression. FTY720-P, an S1P analogue that binds all S1P receptors except S1P 2 , did not inhibit glioblastoma cell migration. Overexpression of S1P 2 further suppressed migration, and blockage of S1P 2 mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S1P 2 receptor stimulation, both Rac1 and RhoA GTPases were activated by S1P treatment in native cells and cells overexpressing S1P 2 . Treatment of U118 cells with the Rhoassociated protein kinase (ROCK) inhibitor Y-27632 restored migration suggesting that ROCK-dependent mechanisms are important. Actin staining of S1P stimulated U118 cells overexpressing B-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S1P 2 overexpression, partially blocked by S1P 1 , or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of S1P inhibition of tumor cell migration via Rho kinase-dependent pathway. (Cancer Res 2005; 65(9): 3788-95)

FoxO proteins are major targets of insulin action. To better define the role of FoxO1 in mediating insulin effects in the liver, we generated liver-specific insulin receptor knockout (LIRKO) and IR/FoxO1 double knockout (LIRFKO) mice. Here we show that LIRKO mice are severely insulin resistant based on glucose, insulin and C-peptide levels, and glucose and insulin tolerance tests, and genetic deletion of hepatic FoxO1 reverses these effects. 13C-glucose and insulin clamp studies indicate that regulation of both hepatic glucose production (HGP) and glucose utilization is impaired in LIRKO mice, and these defects are also restored in LIRFKO mice corresponding to changes in gene expression. We conclude that (1) inhibition of FoxO1 is critical for both direct (hepatic) and indirect effects of insulin on HGP and utilization, and (2) extrahepatic effects of insulin are sufficient to maintain normal whole-body and hepatic glucose metabolism when liver FoxO1 activity is disrupted.

Angiogenesis, or new blood vessel formation, is critical for the growth and spread of tumors. Multiple phases of this process, namely, migration, proliferation, morphogenesis, and vascular stabilization, are needed for optimal tumor growth beyond a diffusion-limited size. The sphingosine 1-phosphate (S1P) receptor-1 (S1P 1 ) is required for stabilization of nascent blood vessels during embryonic development. Here we show that S1P 1 expression is strongly induced in tumor vessels. We developed a multiplex RNA interference technique to downregulate S1P 1 in mice. The small interfering RNA (siRNA) for S1P 1 specifically silenced the cognate transcript in endothelial cells and inhibited endothelial cell migration in vitro and the growth of neovessels into subcutaneous implants of Matrigel in vivo. Local injection of S1P 1 siRNA, but not a negative control siRNA, into established tumors inhibited the expression of S1P 1 polypeptide on neovessels while concomitantly suppressing vascular stabilization and angiogenesis, which resulted in dramatic suppression of tumor growth in vivo. These data suggest that S1P 1 is a critical component of the tumor angiogenic response and argue for the utility of siRNA technology in antiangiogenic therapeutics.