Sphingosine-1-phosphate Axis Research Articles

Lanhuashen stimulates the positive cross-regulation mediated by the S1P axis to ameliorate the disorder of glucolipid metabolism induced by the high sucrose diet in Drosophila melanogaster

Ethnopharmacological relevanceHerba Wanlenbergiae, named ‘Lanhuashen’ (LHS) in Chinese, is derived from the dried herba of Wahlenbergia marginata (Thunb.) A.DC. It is an abundant resource that has been used in traditional Chinese medicine (TCM) for over 600 years. LHS has the effects of enriching consumptive disease and relieving deficient heat, consistent with the therapy for type 2 diabetes mellitus (T2DM) in TCM. As the basic remedy of Yulan Jiangtang capsules, a listed Chinese medicine specifically for treating T2DM, LHS is a potential candidate for an anti-T2DM drug. However, due to the lack of pharmacodynamic studies and chemical component analysis, the application and development of LHS as a treatment for T2DM have been hindered. Aim of the studyTo evaluate the regulation of the disorder of glucolipid metabolism using LHS extracts and its therapeutic potential in T2DM. Materials and methodsChemical components in LHS extracts were analysed using UPLC-Q Exactive-Orbitrap-MS. Subsequently, high sucrose diet (HSD)-induced Drosophila melanogaster were used as suitable models for T2DM in vivo. Behavioural and biochemical tests were performed to evaluate the regulation of the disorder of glucolipid metabolism using LHS in T2DM flies. Furthermore, integrative metabolomic and transcriptomic analysis was applied to reveal the specific effects of LHS extracts on metabolites and genes. Meanwhile, bioinformatic analysis was carried out to predict the targeted transcription factors (TFs) and potentially effective components of LHS extracts. ResultsWe redefined the chemical profile of LHS with 76 identified chemical components, including 65 chemical components for the first time. As indicated by decreased trehalose, glucose and triglyceride levels and increased total protein levels, LHS extracts were perceived to alleviate the disorder of glucolipid metabolism in HSD-induced T2DM fruit flies. Integrative metabolomic and transcriptomic analysis revealed that LHS extracts eliminated the accumulation of sphingolipids and subsequently stimulated the positive cross-regulation mediated by the sphingosine 1-phosphate (S1P) axis, resulting in the activation of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) signalling pathway and inhibition of lysosome-mediated apoptosis. Bioinformatic analysis revealed that the upstream TFs, transcriptional enhancer factor TEF-5 (TEAD3) and peroxisome proliferator-activated receptor alpha (PPARA), were the potential targets of atractylenolide III, dihydrokaempferol and syringaldehyde, the potentially effective components of LHS extracts. Therefore, this TF network was plausibly the basis for the efficacy. ConclusionsLHS extracts broadly modulated TF-dependent gene expression and subsequently stimulated the positive cross-regulation mediated by the S1P axis to ameliorate the disorder of glucolipid metabolism. Our study provides critical evidence considering LHS as a potential drug candidate for T2DM, inspiring the discovery and development of innovative therapeutic agents based on the cross-regulation mediated by the S1P axis for treating T2DM and related complications.

Sex Differences in Sphingosine-1-Phosphate Levels Are Dependent on Ceramide Synthase 1 and Ceramidase in Lung Physiology and Tumor Conditions.

Sex is a biological variable that can reflect clinical outcomes in terms of quality of life, therapy effectiveness, responsiveness and/or toxicity. Sphingosine-1-phosphate (S1P) is a lipidic mediator whose activity can be influenced by sex. To evaluate whether the S1P axis underlies sex 'instructions' in the lung during physiological and oncological lung conditions, sphingosine and S1P were quantified in the blood of healthy (H) volunteers, lung adenocarcinoma (ADK) and squamous cell carcinoma (SCC) patients of both sexes. S1P receptors and their metabolic enzymes were evaluated in the tissues. Circulating levels of S1P were similar among H female and male subjects and female SCC patients. Instead, male and female ADK patients had lower circulating S1P levels. S1P receptor 3 (S1PR3) was physiologically expressed in the lung, but it was overexpressed in male SCC, and female and male ADK, but not in female SCC patients, who showed a significantly reduced ceramide synthase 1 (CERS1) mRNA and an overexpression of the ceramidase (ASAH1) precursor in lung tumor tissues, compared to male SCC and both male and female ADK patients. These findings highlighted sex differences in S1P rheostat in pathological conditions, but not in physiological conditions, identifying S1P as a prognostic mediator depending on lung cancer histotype.

Induction of Inflammation Disrupts the Negative Interplay between STING and S1P Axis That Is Observed during Physiological Conditions in the Lung.

The stimulator of interferon genes (STING) is a master regulator of innate immunity, involved in several inflammatory diseases. Our previous data showed that sphingosine-1-phosphate (S1P) is released during inflammatory conditions in the lung. The aim of this study was to understand the interplay between S1P and STING during both physiological and pathological conditions. The mRNA levels of ceramidase (ASAH1), S1P precursor enzyme, and STING were inversely correlated in healthy lung tissues, but positively correlated in tumor tissues. The activation of STING induced higher expression of ASAH1 and was accompanied by IFN-β and IL-6 release. ASAH1 and sphingosine kinases (SPHK I/II) blockade significantly reduced IL-6, but not IFNβ, after STING activation. In support of this, taking advantage of a mouse model, we found that inflamed lungs had higher levels of inactive ASAH1 when STING was inhibited. This confirmed the human data, where higher levels of STING promoted the activation of ASAH1. Lung cancer patients positive to STING and ASAH1 mRNA levels had a dismal prognosis in that the overall survival was reduced compared to STING/ASAH1 negative patients. These data highlight that during physiological conditions, STING and the S1P axis do not interfere, whereas in lung cancer patients their interplay is associated to poor prognosis.

The Sphingosine 1-Phosphate Axis: an Emerging Therapeutic Opportunity for Endometriosis.

Endometriosis is a common condition in women of reproductive age, but its current interventions are unsatisfactory. Recent research discovered a dysregulation of the sphingosine 1-phosphate (S1P) signaling pathway in endometriosis and showed a positive outcome by targeting it. The S1P axis participates in a series of fundamental pathophysiological processes. This narrative review is trying to expound the reported and putative (due to limited reports in this area for now) interactions between the S1P axis and endometriosis in those pathophysiological processes, to provide some perspectives for future research. In short, S1P signaling pathway is highly activated in the endometriotic lesion. The S1P concentration has a surge in the endometriotic cyst fluid and the peritoneal fluid, with the downstream dysregulation of its receptors. The S1P axis plays an essential role in the migration and activation of the immune cells, fibrosis, angiogenesis, pain-related hyperalgesia, and innervation. S1P receptor (S1PR) modulators showed an impressive therapeutic effect by targeting the different S1P receptors in the endometriosis model, and many other conditions resemble endometriosis. And several of them already got approval for clinical application in many diseases, which means a drug repurposing direction and a rapid clinical translation for endometriosis treatments.

Sphingosine-1-phosphate receptor modulators in stroke treatment.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that can influence a broad range of biological processes through its binding to five distinct G-protein-coupled receptors. S1P receptor modulators are a new group of immunosuppressive agents currently used in the immunotherapy of multiple sclerosis. Inflammation following stroke can exacerbate neuronal injury. Given that S1P signaling is linked to multiple immune processes, therapies targeting the S1P axis may be suitable for treating stroke. In this review, we outline S1P metabolism and S1P receptors, discuss the mechanisms of action of S1P receptor modulators in lymphocyte migration and their direct action on cells of the central nervous system, and provide a concise summary of the efficacy of S1P receptor modulators in animal studies and clinical trials on treatments for stroke.

S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP

Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies (Blood, 2021; Lancet haematology, 2017; Lancet haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients. Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF. Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF. We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner. Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.

S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes.

Background: Adiponectin (Adn), released by adipocytes and other cell types such as skeletal muscle, has insulin-sensitizing and anti-inflammatory properties. Sphingosine 1-phosphate (S1P) is reported to act as effector of diverse biological actions of Adn in different tissues. S1P is a bioactive sphingolipid synthesized by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK) 1 and 2. Consolidated findings support the key role of S1P in the biology of skeletal muscle. Methods and Results: Here we provide experimental evidence that S1P signalling is modulated by globular Adn treatment being able to increase the phosphorylation of SK1/2 as well as the mRNA expression levels of S1P4 in C2C12 myotubes. These findings were confirmed by LC-MS/MS that showed an increase of S1P levels after Adn treatment. Notably, the involvement of S1P axis in Adn action was highlighted since, when SK1 and 2 were inhibited by PF543 and ABC294640 inhibitors, respectively, not only the electrophysiological changes but also the increase of oxygen consumption and of aminoacid levels induced by the hormone, were significantly inhibited. Conclusion: Altogether, these findings show that S1P biosynthesis is necessary for the electrophysiological properties and oxidative metabolism of Adn in skeletal muscle cells.

Sphingosine-1-phosphate in mitochondrial function and metabolic diseases.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite. The past decade has witnessed exponential growth in the field of S1P research, partly attributed to drugs targeting its receptors or kinases. Accumulating evidence indicates that changes in the S1P axis (i.e., S1P production, transport, and receptors) may modify metabolism and eventually mediate metabolic diseases. Dysfunction of the mitochondria on a master monitor of cellular metabolism is considered the leading cause of metabolic diseases, with aberrations typically induced by abnormal biogenesis, respiratory chain complex disorders, reactive oxygen species overproduction, calcium deposition, and mitophagy impairment. Accordingly, we discuss decades of investigation into changes in the S1P axis and how it controls mitochondrial function. Furthermore, we summarize recent scientific advances in disorders associated with the S1P axis and their involvement in the pathogenesis of metabolic diseases in humans, including type 2 diabetes mellitus and cardiovascular disease, from the perspective of mitochondrial function. Finally, we review potential challenges and prospects for S1P axis application to the regulation of mitochondrial function and metabolic diseases; these data may provide theoretical guidance for the treatment of metabolic diseases.

Highlights of This Issue

Aberrant glycosylation is one of the hallmarks of cancer, and cancer cell glycans serve as tumor biomarkers. Glycosylation modulates the functions of glycoproteins; however, the functional significance of protein glycosylation in cancer progression remains poorly defined. Here, β1,6GlcNAc-branched N-glycan–suppressing and N-glycan–defective β4-integrin expressing cell model systems were used to demonstrate that N-glycosylation of β4-integrin contributes to tumor progression. It was determined that β4-integrin clustering by galectin-3–mediated cross-linking of β1,6GlcNAc-branched N-glycans on β4-integrin activates PI3K/Akt signaling, thereby promoting cancer cell behavior. Thus, N-glycosylation of β4-integrin represents a potential therapeutic target for cancer.Adaptive cellular responses induced by cancer therapeutics are emerging as an important mechanism for acquired resistance to chemotherapies. In this study, Fang and colleagues identified olaparib-induced FOXM1 expression as an adaptive response that induces the expression of homologous recombination (HR) repair genes. Knockdown of olaparib-induced FOXM1 expression by RNAi or pharmacologic inhibition by thiostrepton downregulates the expression of HR repair genes, increases DNA damage and PARP1 trapping, and enhances sensitivity to olaparib. This study highlights the important role of FOXM1 in the adaptive response to olaparib, and that targeting the adaptive response mediated by FOXM1 extends the efficacy of PARP inhibitors.A major problem that limits the effectiveness of cancer chemotherapeutics is the development of drug resistance. Recently, dysregulation of the Hippo pathway core component LATS has been implicated in this process, but its underlying mechanisms remain unknown. Now, Yeung and colleagues functionally characterize a novel signaling pathway in which anti-tubulin drugs activate Cdk1 and phosphorylate LATS, which then binds prolyl isomerase Pin1 and inhibits its anti-apoptotic function. Importantly, loss of LATS phosphorylation reduces taxol-induced cell death. Together, this work provides new insight into the anti-tubulin drug response and demonstrates the Cdk1-LATS-Pin1 signaling axis as a promising target for treatment.ATP-binding cassette (ABC) transporters are a known mechanism of drug resistance, however multiple clinical trials targeting ABCB1 have failed. Thus, it is hypothesized here, that ABC transporters aggravate cancer biology by exporting endogenous lipid mediator sphingosine-1-phosphate (S1P) regardless of drugs. S1P generated by SphK1 and exported via ABCC1 aggravated cancer in a malicious feed-forward manner by amplifying the S1P axis in vitro, and worsened cancer progression in vivo. Patients with high levels of both SphK1 and ABCC1 have worse survival. While, ABCB1, which does not export S1P, did not demonstrate these findings. This suggests a critical role for the S1P axis in breast cancer progression.

Abstract P5-03-05: Sphingosine-1-phosphate produced by sphingosine kinase 1 and exported via ABCC1 shortens survival of mice and humans with breast cancer

Abstract Background: Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator that is generated by sphingosine kinase 1 (SphK1) when it is phosphorylated (pSphK1) inside cells, has been implicated in regulation of many process important for breast cancer progression. Previously we have shown that S1P is exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathological consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, we report that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients via exporting S1P. Materials and methods: Microarray based gene expression data of 2509 patients associated with their survival were obtained from METABRIC database. Single gene survival analysis based on expressin of SphK1, and dual ABCC1 or ABCB1 and SphK1 survival analyses were perfomerd. For protein analyses, tissues were obrained from 275 patients with stage 1-3 breast cancers treated in Yokohama City University Medical Center in Japan between 2006 and 2008. The expression of pSphK1 was analyzed by immunohistochemistry and investigate the relationship with clinicopathological findings. For in vitro and in vivo experiments, breast cancer cell lines were transfected by ABCB1, ABCC1 or vector transiently or stably. BALB/c nu/nu mice and BALB/c mice were used for in vivo experiments. S1P was measured by LC-ESI-MS/MS. Results: SphK1 expression significantly associate with worse overall survival (median survival of 124 months with high SphK1 expression compared to 163 months for patients with low SphK1 expression, p=0.0014). Although patients with high ABCC1 expression had only a slightly worse overall survival of 150 months, those with high levels of both SphK1 and ABCC1 had much worse prognosis with median overall survival of 114 months (p < 0.0068). Such association was not observed with ABCB1 expression. The frequency of strong pSphK1 protein expression was higher in HER2 enrhiched or TNBC than in Luminal. pSphK1 was more prevalent and increased in a larger tumors and in tumors from patients with lymph node metastases. Patients with breast cancers that express both pSphK1 and ABCC1 proteins have significantly shorter disease free survival. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 cells enhanced S1P secretion, proliferation and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary pad markedly enhanced tumor growth, angiogenesis and lymphangiogenesis with concomitant increases in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of SphK1 and its own formation. Conclusions: Our findings suggest that production and export of S1P via ABCC1, but not ABCB1, is associated with worse overall and disease free survival of breast cancer patients and that S1P axis play a role in aggressive biology of breast cancer progression and metastasis. Citation Format: Yamada A, Nagahashi M, Aoyagi T, Huang W-C, Lima S, Miyazaki H, Narui K, Ishikawa T, Endo I, Waters MR, Milstien S, Spiegel S, Takabe K. Sphingosine-1-phosphate produced by sphingosine kinase 1 and exported via ABCC1 shortens survival of mice and humans with breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-05.

Sphingosine kinase 1 in breast cancer.

Breast cancer affects 1 out of 8 women in the US and is the second highest cause of death from cancer for women, leading to considerable research examining the causes, progression, and treatment of breast cancer. Over the last two decades, sphingosine-1-phosphate (S1P), a potent sphingolipid metabolite, has been implicated in many processes important for breast cancer including growth, progression, transformation and metastasis, and is the focus of this review. In particular, one of the kinases that produces S1P, sphingosine kinase 1 (SphK1), has come under increasing scrutiny as it is commonly upregulated in breast cancer cells and has been linked with poorer prognosis and progression, possibly leading to resistance to certain anti-cancer therapies. In this review, we will also discuss preclinical studies of both estrogen receptor (ER) positive as well as triple-negative breast cancer mouse models with inhibitors of SphK1 and other compounds that target the S1P axis and have shown good promise in reducing tumor growth and metastasis. It is hoped that in the future this will lead to development of novel combination approaches for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

Sphingosine 1-Phosphate Signaling as a Target in Hepatic Fibrosis Therapy.

Liver fibrosis is an excess production of extracellular matrix proteins as a result of chronic liver disease which leads to cell death and organ dysfunction. The key cells involved in fibrogenesis are resident hepatic stellate cells (HSCs) which are termed myofibroblasts after activation, acquiring contractile, proliferative, migratory and secretory capability. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with well-established effects on angiogenesis, carcinogenesis and immunity. Accumulating evidence demonstrates that this metabolite is involved in the profibrotic inflammatory process through the regulation of pleiotropic cell responses, such as vascular permeability, leukocyte infiltration, cell survival, migration, proliferation and HSCs differentiation to myofibroblasts. S1P is synthesized by sphingosine kinases (SphKs) and many of its actions are mediated by S1P specific cell surface receptors (S1P1-5), although different intracellular targets of S1P have been identified. Modulation of SphKs/S1P/S1P receptors signaling is known to result in beneficial effects on various in vivo and in vitro models of liver fibrosis. Thus, a better knowledge of the molecular mechanisms involved in the modulation of the S1P pathway could help to improve liver fibrosis therapy. In this review, we analyze the effects of the S1P axis on the fibrogenic process, and the involvement of a range of inhibitors or approaches targeting enzymes related to S1P in the abrogation of pathological fibrogenesis. All in all, targeting this pathway offers therapeutic potential in the treatment of hepatic fibrosis.

Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway: Therapeutic Targets in Autoimmunity and Inflammation.

Sphingosine-1-phosphate (S1P) and S1P receptors (S1PR) are ubiquitously expressed. S1P-S1PR signaling has been well characterized in immune trafficking and activation in innate and adaptive immune systems. However, the full extent of its involvement in the pathogenesis of autoimmune diseases is not well understood. FTY720 (fingolimod), a non-selective S1PR modulator, significantly decreased annualized relapse rates in relapsing-remitting multiple sclerosis (MS). FTY720, which primarily targets S1P receptor 1 asafunctional antagonist, arrests lymphocyte egress from secondary lymphoid tissues and reduces neuroinflammation in the central nervous system (CNS). Recent studies suggest that FTY720 also decreases astrogliosis and promotes oligodendrocyte differentiation within the CNS and may have therapeutic benefit to prevent brain atrophy. Since S1P signaling is involved in multiple immune functions, therapies targeting S1P axis may be applicable to treat autoimmune diseases other than MS. Currently, over a dozen selective S1PR and S1P pathway modulators with potentially superior therapeutic efficacy and better side-effect profiles are in the pipeline of drug development. Furthermore, newly characterized molecules such as apolipoprotein M (ApoM) (S1P chaperon) and SPNS2 (S1P transporter) are also potential targets for treatment of autoimmune diseases. Finally, the application of therapies targeting S1P and S1P signaling pathways may be expanded to treat several other immune-mediated disorders (such as post-infectious diseases, post-stroke and post-stroke dementia) and inflammatory conditions beyond their application in primary autoimmune diseases.

Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma.

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability.

Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts.

The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative roleon CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions. • Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB. • H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB. • CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB. • S1P1/3 were involved on CM-induced morphogenesis and migration of MAB. • Matrigel plug assay showed the role of S1P axis in MAB-endothelial cell interaction.

Abstract 1890: Activated SphK1 and export of S1P via ABCC1 shorten disease free survival in breast cancer

Abstract ATP-binding cassette (ABC) transporters are known to be multidrug resistance proteins that efflux various compounds out of cells including chemotherapeutic agents. A number of clinical trials have been conducted targeting ABCB1: however, none showed beneficial effects. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P), which is generated by sphingosine kinase 1 (SphK1) inside breast cancer cells and exerts its functions by binding to its specific cell surface G-protein coupled receptors (S1PR1-5) after being exported, is now known as a key regulatory molecule in breast cancer progression. We have previously demonstrated that ABCC1 and ABCG2, but not ABCB1 export S1P out of MCF7 human breast cancer cells. We hypothesized that ABCC1 expression in the presence of S1P produced by activated SphK1 in human breast cancer is associated with poor prognosis. We demonstrate the role of ABCC1 and SphK1/ S1P axis in breast cancer progression utilizing ABC transporter transduced breast cancer cell lines for in vitro and in vivo study. For clinical study, we analyze the expression of ABC transporters and S1P signaling factors utilizing a tissue microarray constructed from 276 breast cancer samples. Overexpression of ABCC1 in breast cancer cell lines export more S1P. ABCC1 overexpression but not ABCB1 enhance proliferation and cell migration, and promote angiogenesis (AG) and lymph angiogenesis (LAG). MK571 which is inhibitor of ABCC1 suppress them. Overexpression of ABCC1 also aggravate tumor grow and contribute poor survival in syngeneic and xenograft mice model. In the clinical study, ABCC1 expression was associated with significantly shorter disease free survival (DFS) while ABCB1 was not. pSphK1 high expression group had significantly shorter DFS. Most of the patients expressed S1PR1; however, there were no significant differences in prognosis. Patients with tumors expressing both pSphK1 and ABCC1 had significantly shorter DFS, while patients expressing both ABCB1 and pSphK1 did not. These translational findings suggest that inside-out signaling of S1P via ABCC1 may play a significant role in the course of human breast cancer progression. This work was supported by grants from the National Institute of Health (R01CA160688 to K.T and R01CA61774 to S.S). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. Citation Format: Akimitsu Yamada, Masayuki Nagahashi, Tomoyoshi Aoyagi, Wei C. Huang, Krista P. Terracina, Jeremy C. Allegood, Santiago Lima, Sheldon Milstien, Sarah Spiegel, Kumiko Kida, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. Activated SphK1 and export of S1P via ABCC1 shorten disease free survival in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1890. doi:10.1158/1538-7445.AM2014-1890

B26 Targeting Sphingosine-1-phosphate Metabolism/axis May Be Beneficial In Huntington Disease Models

Background Sphingosine-1-phosphate (S1P) is a potent signalling sphingolipid that acts as a high affinity agonist at five known G protein-coupled receptors, S1PR1-S1PR5, and regulates brain cell homeostasis and survival. S1P metabolism is quite complex and involves the action of different enzymes. S1P is synthesised by sphingosine kinase-1 and -2 (SPHK1 and 2) and degraded either by S1P-phosphate (SPP) or by S1P-lyase (SGPL1). Recent evidence indicates that pharmacological modulation of S1P metabolism/axis may represent a promising therapeutic approach for multiple disorders including neurodegenerative diseases. Aims Here, we aim to explore whether S1P metabolism/signalling pathway may have the potential to be molecularly targeted for developing new therapeutic strategies for the treatment of HD. Methods In vitro experiments were performed in mouse striatal-derived cells expressing wild-type (SThdh7/7) or mutant huntingtin (SThdh111/111). In vivo experiments were carried out in symptomatic R6/2 and YAC128 HD mouse models. Results Our findings reveal an overall aberrant expression of S1P metabolising enzymes both in HD cells and mouse brain tissues with increased levels of SGPL1, which might likely correlate with its increased activity and subsequent alteration of S1P bio-availability. Also, we found that targeting specific molecules in S1P/metabolism/axis activates pro-survival pathways in SThdh111/111 cells. Conclusion These results support the hypothesis of an aberrant metabolism of S1P in HD and indicate that the modulation of its signalling pathway may be beneficial. Thus, we believe S1P axis has the potential of providing the possibility to develop new therapeutic interventions for treating HD. This research is supported by Marie Curie International Incoming Fellowship (PIIF-GA-2011–300197) granted to V. M. within the 7th European Community Framework Programme.

Abstract P2-05-06: Does the lipid mediator sphingosine-1-phosphate have a role in obesity-related cancer progression?

Abstract INTRODUCTION: Obesity, which is the number one health risk in US ranked by the CDC, is an established independent prognostic factor for breast cancer patients. While the link between obesity and elevated breast cancer mortality is well known, the underlying mechanisms are poorly understood. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression and inflammation. S1P is generated by two sphingosine kinases, SphK1 and SphK2, and exerts its functions by binding to specific G protein-coupled receptors (S1PR1-5). We have recently shown that SphK1, but not SphK2, produces S1P that is exported from breast cancer cells, mediated by the ATP-binding cassette transporter, ABCC1. Furthermore, we discovered that another S1P transporter, Spns2, is important for the lymphatic network formation in the LNs. FTY720, which after phosphorylation is a S1PR1 functional antagonist, was recently approved by FDA for multiple sclerosis. FTY720 also has been suggested to have some anti-cancer actions. Our hypothesis is that obesity up-regulates SphK1, which produces more S1P; the elevated levels of S1P in both tumor and its microenvironment stimulate breast cancer progression. FTY720 is expected to disrupt the SphK1/S1P/S1PR1 axis, which is strengthened by obesity, and to reduce cancer metastasis and prolong survival. We tested our hypothesis by utilizing animal models of breast cancer with obesity treated with FTY720. METHODS: We utilized two different syngeneic breast cancer mouse models: 4T1-luc2 cells in BALB/c mice and E0771 cells in C57Bl/6 mice, both inoculated into mammary fat pads of mice fed with normal or high fat diet. FTY720 was given orally. Western blot, QPCR and LC-ESI-MS/MS assays were used. RESULTS: We observed that breast tumors in obese animals expressed higher levels of SphK1 and S1P transporters, such as ABCC1 and Spns2, as compared to animals on a normal diet. The levels of S1P in the plasma of obese mice were also elevated, which appears to be a consequence of the higher production of S1P by SphK1 in the tumor and its microenvironment. Importantly, tumor progression in both models was suppressed significantly by administration of FTY720. Interestingly, the cancer progression was suppressed more efficiently in the obese mice than the lean mice. Further, LC-ESI-MS/MS analysis showed that FTY720 suppressed S1P levels not only in the blood, but also in the tumor interstitial fluid. CONCLUSIONS: These results suggest that the S1P axis is strengthened by obesity, and has a role in cancer progression. Targeting the S1P axis with FTY720 may be useful for treating breast cancer in individuals with obesity. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and R01CA160688 (to K.T.). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-06.

Role of sphingosine 1-phosphate in trafficking and mobilization of hematopoietic stem cells

The 'mobilization' of hematopoietic stem cells (HSCs) out of the bone marrow and into the peripheral blood is used clinically to obtain HSCs for transplantation. Although generally successful, mobilization protocols remain imperfect and the mechanisms involved are not fully understood. This review discusses the latest findings in respect to the mechanisms involved in the egress of HSCs from the bone marrow into the circulation and the potential for these recent developments to improve mobilization procedures. It has recently become apparent that the bioactive lipid sphingosine 1-phosphate (S1P) plays an active role in attracting HSCs into the peripheral blood. S1P is the first factor identified that provides a chemoattractant gradient promoting the movement of HSCs into the peripheral blood. Drugs that mimic S1P are available with others in development, raising the possibility of increasing the strength of the egress signal and thereby improving the efficacy of mobilization procedures. S1P is the first egress factor described for HSCs, but the details of the underlying biology are only just emerging. Although manipulating the S1P axis to enhance mobilization protocols is an exciting possibility, much needs to be learned before improvements in mobilization strategies can be realized.

The Apolipoprotein M–Sphingosine-1-Phosphate Axis: Biological Relevance in Lipoprotein Metabolism, Lipid Disorders and Atherosclerosis

Apolipoprotein M (apoM) is a plasma apolipoprotein that mainly associates with high-density lipoproteins. Hence, most studies on apoM so far have investigated its effect on and association with lipid metabolism and atherosclerosis. The insight into apoM biology recently took a major turn. ApoM was identified as a carrier of the bioactive lipid sphingosine-1-phosphate (S1P). S1P activates five different G-protein-coupled receptors, known as the S1P-receptors 1–5 and, hence, affects a wide range of biological processes, such as lymphocyte trafficking, angiogenesis, wound repair and even virus suppression and cancer. The ability of apoM to bind S1P is due to a lipophilic binding pocket within the lipocalin structure of the apoM molecule. Mice overexpressing apoM have increased plasma S1P concentrations, whereas apoM-deficient mice have decreased S1P levels. ApoM-S1P is able to activate the S1P-receptor-1, affecting the function of endothelial cells, and apoM-deficient mice display impaired endothelial permeability in the lung. This review will focus on the putative biological roles of the new apoM–S1P axis in relation to lipoprotein metabolism, lipid disorders and atherosclerosis.