Babak Oskouian

Sphingosine-1-phosphate lyase potentiates apoptosis via p53- and p38-dependent pathways and is down-regulated in colon cancer

Sphingolipid metabolites such as sphingosine-1-phosphate (S1P) and ceramide modulate apoptosis during development and in response to stress. In general, ceramide promotes apoptosis, whereas S1P stimulates cell proliferation and protects against apoptosis. S1P is irreversibly degraded by the enzyme S1P lyase (SPL). In this study, we show a crucial role for SPL in mediating cellular responses to stress. SPL expression in HEK293 cells potentiated apoptosis in response to stressful stimuli including DNA damage. This effect seemed to be independent of ceramide generation but required SPL enzymatic activity and the actions of p38 MAP kinase, p53, p53-inducible death domain protein (PIDD), and caspase-2 as shown by molecular and chemical inhibition of each of these targets. Further, SPL expression led to constitutive activation of p38. Endogenous SPL expression was induced by DNA damage in WT cells, whereas SPL knockdown diminished apoptotic responses. Importantly, SPL expression was significantly down-regulated in human colon cancer tissues in comparison with normal adjacent tissues, as determined by quantitative real-time PCR (Q-PCR) and immunohistochemical analysis. Down-regulation of S1P phosphatases was also observed, suggesting that colon cancer cells manifest a block in S1P catabolism. In addition, SPL expression and activity were down-regulated in adenomatous lesions of the Min mouse model of intestinal tumorigenesis. Taken together, these results indicate that endogenous SPL may play a physiological role in stress-induced apoptosis and provide an example of altered SPL expression in a human tumor. Our findings suggest that genetic or epigenetic changes affecting intestinal S1P metabolism may correlate with and potentially contribute to carcinogenesis.

The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.

Cancer Treatment Strategies Targeting Sphingolipid Metabolism

Ceramide and sphingosine-1-phosphate are related sphingolipid metabolites that can be generated through a de novo biosynthetic route or derived from the recycling of membrane sphingomyelin. Both these lipids regulate cellular responses to stress, with generally opposing effects. Sphingosine-1-phosphate functions as a growth and survival factor, acting as a ligand for a family of G protein-coupled receptors, whereas ceramide activates intrinsic and extrinsic apoptotic pathways through receptor-independent mechanisms. A growing body of evidence has implicated ceramide, sphingosine-1-phosphate and the genes involved in their synthesis, catabolism and signaling in various aspects of oncogenesis, cancer progression and drug- and radiation resistance. This may be explained in part by the finding that both lipids impinge upon the PI3K/ AKT pathway, which represses apoptosis and autophagy. In addition, sphingolipids influence cell cycle progression, telomerase function, cell migration and stem cell biology. Considering the central role of ceramide in mediating physiological as well as pharmacologically stimulated apoptosis, ceramide can be considered a tumor-suppressor lipid. In contrast, sphingosine-1-phosphate can be considered a tumor-promoting lipid, and the enzyme responsible for its synthesis functions as an oncogene. Not surprisingly, genetic mutations that result in reduced ceramide generation, increased sphingosine-1-phosphate synthesis or which reduce steady state ceramide levels and increase sphingosine-1-phosphate levels have been identified as mechanisms of tumor progression and drug resistance in cancer cells. Pharmacological tools for modulating sphingolipid pathways are being developed and represent novel therapeutic strategies for the treatment of cancer.

Protection of LPS-Induced Murine Acute Lung Injury by Sphingosine-1-Phosphate Lyase Suppression

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Sphingosine-1-Phosphate Metabolism and Intestinal Tumorigenesis: Lipid Signaling Strikes Again

Sphingolipids are an evolutionary conserved class of membrane lipids synthesized by all eukaryotic cells. The biological functions of sphingolipids are diverse, encompassing structural roles through their participation in membrane lipid rafts, and informational roles via the involvement of their metabolites in signal transduction pathways. An important sphingolipid metabolite is sphingosine-1-phosphate (S1P), which acts through G protein-coupled receptors present on mammalian cells, thereby stimulating cell proliferation, angiogenesis and inhibiting apoptosis. The main enzyme responsible for S1P synthesis, sphingosine kinase 1 (Sphk1), behaves as an oncogene in experimental systems and is required for polyp enlargement in the Min mouse model of intestinal tumorigenesis. S1P is irreversibly degraded by S1P lyase (SPL), an enzyme that is highly expressed in enterocytes, where it is involved in metabolism of dietary sphingolipids. Forced expression of SPL sensitizes human cells to various stressful stimuli and enhances apoptotic cell death. SPL expression is induced in response to DNA damaging agents in a time- and concentration-dependent manner. On the other hand, SPL is downregulated in human colon cancers and in Min mouse adenomas compared to adjacent uninvolved tissues. These observations suggest that SPL, like Sphk1, may play a role in tumorigenesis. Added support for this notion comes from the fact that S1P-specific antibodies slow tumor progression and angiogenesis in murine xenograft and allograft models. Together, these recent studies have established a link between S1P signaling, metabolism and carcinogenesis that may have implications regarding colon cancer screening, dietary chemoprevention and therapeutics.

Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/ STAT3 signaling pathway

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Overexpression of fatty acid synthase in SKBR3 breast cancer cell line is mediated via a transcriptional mechanism

Overexpression of fatty acid synthase (FAS) in certain breast, prostate and ovarian tumors has been correlated with aggressive cancer phenotype and poor prognosis. The objective of this study was to use a breast cancer-derived cell line, SKBR3, as a model to define the underlying mechanism for overexpression of FAS in cancer cells. Different stages of gene expression where overproduction of FAS could potentially be achieved were investigated. Whereas gross chromosomal rearrangement at the FAS locus, amplification of the FAS gene, increases in FAS message stability and longer half-life of the FAS protein were not detected, an increase in the rate of transcription of the FAS gene, and consequently a higher abundance of FAS-mRNA, was found to be primarily responsible for FAS overexpression in this cell line.

Natural Sphingadienes Inhibit Akt-Dependent Signaling and Prevent Intestinal Tumorigenesis

Sphingolipid metabolites regulate cell proliferation, migration, and stress responses. Alterations in sphingolipid metabolism have been proposed to contribute to carcinogenesis, cancer progression, and drug resistance. We identified a family of natural sphingolipids called sphingadienes and investigated their effects in colon cancer. We find that sphingadienes induce colon cancer cell death in vitro and prevent intestinal tumorigenesis in vivo. Sphingadienes exert their influence by blocking Akt translocation from the cytosol to the membrane, thereby inhibiting protein translation and promoting apoptosis and autophagy. Sphingadienes are orally available, are slowly metabolized through the sphingolipid degradative pathway, and show limited short-term toxicity. Thus, sphingadienes represent a new class of therapeutic and/or chemopreventive agents that blocks Akt signaling in neoplastic and preneoplastic cells.

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1&Dgr; yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

YAP1 confers resistance to the fatty acid synthase inhibitor cerulenin through the transporter Flr1p in Saccharomyces cerevisiae

Abstract In this study, we utilized a genetic approach to identify genes which render yeast cells resistant to cerulenin (Cer), a potent and noncompetitive inhibitor of fatty acid synthase (FAS). Overexpression of the yeast transcription factor Yap1p was found to confer Cer resistance (CerR). This resistance was shown to be less pronounced in a strain deleted for YCF1, a multidrug resistance ABC transporter, supporting previous observations that implicated YCF1 in mediating CerR. However, isolation of YAP1 as a high-copy CerR gene in a ycf1Δ strain suggested that YAP1-induced CerR was mediated by additional downstream effectors. Overexpression of neither glutathione reductase nor a predicted aryl alcohol dehydrogenase (the products of two YAP1-regulated genes involved in detoxification) conferred CerR. Overexpression of ATR1, another YAP1-regulated gene previously implicated in conferring resistance to a number of cytotoxic drugs, was also incapable of making cells resistant to Cer. In contrast, overexpression of Flr1p, a yeast transporter of the major facilitator superfamily which is also under the control of YAP1, was sufficient to confer CerR in an otherwise wild-type background. Moreover, CerR was markedly diminished in a strain deleted for FLR1. These findings implicate members of both of the transporter superfamilies involved in multiple drug resistance (MDR) in the acquisition of CerR in yeast. Furthermore, our studies indicate that yeast may be a useful model system in which to investigate the role of FAS in cancer biology and the effects of Cer on eukaryotic cell growth.