Yusuf A. Hannun

Biologically active sphingolipids in cancer pathogenesis and treatment

Biologically active sphingolipids have key roles in the regulation of several fundamental biological processes that are integral to cancer pathogenesis. Recent significant progress in understanding biologically active sphingolipid synthesis, specifically within ceramide and sphingosine-1-phosphate (S1P)-mediated pathways, has identified crucial roles for these molecules both in cancer development and progression. Ceramide — a central molecule in sphingolipid metabolism — in effect functions as a tumour-suppressor lipid, inducing antiproliferative and apoptotic responses in various cancer cells. Conversely, S1P induces responses that, on aggregate, render S1P a tumour-promoting lipid. These discoveries are paving the way for the advancement of anticancer therapies.

Bioactive sphingolipids: metabolism and function

Sphingolipids (SLs) are essential constituents of eukaryotic cells. Besides playing structural roles in cellular membranes, some metabolites, including ceramide, sphingosine, and sphingosine-1-phosphate, have drawn attention as bioactive signaling molecules involved in the regulation of cell growth, differentiation, senescence, and apoptosis. Understanding the many cell regulatory functions of SL metabolites requires an advanced knowledge of how and where in the cell they are generated, converted, or degraded. This review will provide a short overview of the metabolism, localization, and compartmentalization of SLs. Also, a discussion on bioactive members of the SL family and inducers of SL enzymes that lead to ceramide generation will be presented.

The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-α

In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor‐α (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase‐2 (COX‐2) induction as early as 2 h. This TNF‐induced PGE2 production was inhibited by NS398, a COX‐2 selective inhibitor. GC‐MS analysis revealed that only COX‐2‐generated prostanoids were produced in response to TNF, thus providing further evidence of COX‐2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX‐2 induction and PGE2 production was evaluated. Sphingosine‐1‐phosphate (S1P) induced both COX‐2 and PGE2 in a dose‐responsive manner with an apparent ED50 of 100–300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5‐fold increase in sphingosine‐1‐phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF‐induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX‐2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX‐2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphin‐gosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX‐2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.—Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., Hannun, Y A. The sphingosine kinase 1/sphingosine‐1‐phosphate pathway mediates COX‐2 induction and PGE2 production in response to TNF‐α, FASEB J., 17, 1411–1421 (2003)

(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-Derythro-2-(N-myristoylamino)-1-phenyl-1-propanol (Derythro-MAPP). Addition of 5 μMD-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G/G phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceramidase to an extent similar to that seen with C-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC of 1-5 μM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC > 500 μM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.

Substantial contribution of extrinsic risk factors to cancer development

Recent research has highlighted a strong correlation between tissue-specific cancer risk and the lifetime number of tissue-specific stem-cell divisions. Whether such correlation implies a high unavoidable intrinsic cancer risk has become a key public health debate with the dissemination of the ‘bad luck’ hypothesis. Here we provide evidence that intrinsic risk factors contribute only modestly (less than ~10–30% of lifetime risk) to cancer development. First, we demonstrate that the correlation between stem-cell division and cancer risk does not distinguish between the effects of intrinsic and extrinsic factors. We then show that intrinsic risk is better estimated by the lower bound risk controlling for total stem-cell divisions. Finally, we show that the rates of endogenous mutation accumulation by intrinsic processes are not sufficient to account for the observed cancer risks. Collectively, we conclude that cancer risk is heavily influenced by extrinsic factors. These results are important for strategizing cancer prevention, research and public health.

Ceramide Kinase Mediates Cytokine- and Calcium Ionophore-induced Arachidonic Acid Release

Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A2, the initial rate-limiting step. In this study, ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1β (IL-1β), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, ceramide kinase, was activated in response to IL-1β and A23187. RNA interference targeted to ceramide kinase specifically down-regulated ceramide kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1β and A23187. Down-regulation of ceramide kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that ceramide kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A2 activation. This study identifies previously unknown roles for ceramide kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.

Simulation and validation of modelled sphingolipid metabolism in Saccharomyces cerevisiae

Mathematical models have become a necessary tool for organizing the rapidly increasing amounts of large-scale data on biochemical pathways and for advanced evaluation of their structure and regulation. Most of these models have addressed specific pathways using either stoichiometric or flux-balance analysis, or fully kinetic Michaelis–Menten representations, metabolic control analysis, or biochemical systems theory. So far, the predictions of kinetic models have rarely been tested using direct experimentation. Here, we validate experimentally a biochemical systems theoretical model of sphingolipid metabolism in yeast. Simulations of metabolic fluxes, enzyme deletion and the effects of inositol (a key regulator of phospholipid metabolism) led to predictions that show significant concordance with experimental results generated post hoc. The model also allowed the simulation of the effects of acute perturbations in fatty-acid precursors of sphingolipids, a situation that is not amenable to direct experimentation. The results demonstrate that modelling now allows testable predictions as well as the design and evaluation of hypothetical ‘thought experiments’ that may generate new metabolomic approaches.

Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C14-C26 N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (Cn-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C16-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C16-LacCer (10.0 µM) and the abundant HexCer was C24-HexCer (2.5 µM). The abundant Cer, C24-Cer (4.0 µM), was not influenced by fasting; however, levels of C16-C20 Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL3 was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis.

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 resistant11-1 (fbr11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B1 (FB1), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base1 (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB1. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1-phosphate in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.

Loss of sphingosine kinase-1 activates the intrinsic pathway of programmed cell death: modulation of sphingolipid levels and the induction of apoptosis

Activation of sphingosine kinase‐1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti‐apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down‐regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF‐7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546‐20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF‐7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.