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Background and Purpose: Sphingosine kinase 1 catalyses formation of the bioactive lipid, sphingosine 1-phosphate, which protects cancer cells from apoptosis. Therefore, sphingosine kinase 1 is a novel target for intervention with anti-cancer agents. We have assessed the effect of the anti-cancer agent, resveratrol and its dimers (ampelopsin A and balanocarpol) on sphingosine kinase 1 activity and on survival of MCF-7 breast cancer cells.Experimental Approach: Ampelopsin A and balanocarpol were purified from Hopea dryobalanoides and their effect on sphingosine kinase 1 activity and expression, [(3)H] thymidine incorporation, ERK-1/2 phosphorylation and PARP activity assessed in MCF-7 cells.Key Results: Resveratrol, ampelopsin A and balanocarpol were novel inhibitors of sphingosine kinase 1 activity. Balanocarpol was a mixed inhibitor (with sphingosine) of sphingosine kinase 1 with a K(ic) = 90 ± 10 µM and a K(iu) of ∼500 µM. Balanocarpol and ampelopsin A also induced down-regulation of sphingosine kinase 1 expression and reduced DNA synthesis, while balanocarpol stimulated PARP cleavage in MCF-7 breast cancer cells. Resveratrol was a competitive inhibitor (with sphingosine) of sphingosine kinase 1 with a K(ic) = 160 ± 40 µM, reduced sphingosine kinase 1 expression and induced PARP cleavage in MCF-7 cells.Conclusions and Implications: Each molecule of balanocarpol may bind at least two sphingosine kinase 1 catalytic molecules to reduce the activity of each simultaneously. These findings suggest that resveratrol, ampelopsin A and balanocarpol could perturb sphingosine kinase 1-mediated signalling and this might explain their activity against MCF-7 breast cancer cells. [ABSTRACT FROM AUTHOR]

Background: We previously reported that sphingosine 1-phosphate receptor 4 (S1P(4)) is expressed and stimulates the ERK-1/2 pathway via a human epidermal growth factor receptor 2 (HER2)-dependent mechanism in oestrogen receptor-negative (ER(-)) MDA-MB-453 breast cancer cells.Methods: Clinical relevance of S1P(4) and sphingosine kinase 1 (SK1, which catalyses the formation of S1P) was assessed in a cohort of 140 ER(-) breast tumours by immunohistochemistry (IHC) and the weighted histoscore method. Additional evidence for a functional interaction between S1P(4) and SK1 and between HER2 and SK1 was obtained using MDA-MB-453 cells.Results: High S1P(4) expression is associated with shorter disease-free (P=0.014) and disease-specific survival (P=0.004), and was independent on multivariate analysis. In addition, patients with tumours that contain high and low levels of SK1 and S1P(4), respectively, have a significantly shorter disease-free survival (P=0.043) and disease-specific survival (P=0.033) compared with patients whose tumours contain both low S1P(4) and SK1 levels. In addition, high tumour expression of SK1 was significantly associated with shorter disease-specific survival (P=0.0001) in patients with HER2-positive tumours. Treatment of MDA-MB-453 cells with the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) reduced the basal and S1P/S1P(4)-induced activation of ERK-1/2 and altered HER2 trafficking in these cells.Conclusion: These findings highlight an important role for S1P(4) and SK1 in ER(-) breast cancer progression. [ABSTRACT FROM AUTHOR]

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemic stage in which B-cell precursors display self-renewal ability, initiating leukemia resembling PAX5 P80R or Ph-like human B-ALL. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are potential treatment avenues for IL-7R-related cases. Our model, a resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy., (© 2021. The Author(s).)

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15542. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Dihydroceramide desaturase (Degs1) catalyses the introduction of a 4,5-trans double bond into dihydroceramide to form ceramide. We show here that Degs1 is polyubiquitinated in response to retinol derivatives, phenolic compounds or anti-oxidants in HEK293T cells. The functional predominance of native versus polyubiquitinated forms of Degs1 appears to govern cytotoxicity. Therefore, 4-HPR or celecoxib appear to stimulate the de novo ceramide pathway (with the exception of C24:0 ceramide), using native Degs1, and thereby promote PARP cleavage and LC3B-I/II processing (autophagy/apoptosis). The ubiquitin-proteasomal degradation of Degs1 is positively linked to cell survival via XBP-1s and results in a concomitant increase in dihydroceramides and a decrease in C24:0 ceramide levels. However, in the case of 4-HPR or celecoxib, the native form of Degs1 functionally predominates, such that the apoptotic programme is sustained. In contrast, 4-HPA or AM404 do not produce apoptotic ceramide, using native Degs1, but do promote a rectifier function to induce ubiquitin-proteasomal degradation of Degs1 and are not cytotoxic. Therefore, Degs1 appears to function both as an 'inducer' and 'rectifier' of apoptosis in response to chemical cellular stress, the dynamic balance for which is dependent on the nature of chemical stress, thereby determining cytotoxicity. The de novo synthesis of ceramide or the ubiquitin-proteasomal degradation of Degs1 in response to anti-oxidants, retinol derivatives and phenolic compounds appear to involve sensors, and for rectifier function, this might be Degs1 itself., (© 2021. The Author(s).)

The translocation of sphingosine kinase 1 (SK1) to the plasma membrane (PM) is crucial in promoting oncogenesis. We have previously proposed that SK1 exists as both a monomer and dimer in equilibrium, although it is unclear whether these species translocate to the PM via the same or different mechanisms. We therefore investigated the structural determinants involved to better understand how translocation might potentially be targeted for therapeutic intervention. We report here that monomeric WT mouse SK1 (GFP-mSK1) translocates to the PM of MCF-7L cells stimulated with carbachol or phorbol 12-myristate 13-acetate, whereas the dimer translocates to the PM in response to sphingosine-1-phosphate; thus, the equilibrium between the monomer and dimer is sensitive to cellular stimulus. In addition, carbachol and phorbol 12-myristate 13-acetate induced translocation of monomeric GFP-mSK1 to lamellipodia, whereas sphingosine-1-phosphate induced translocation of dimeric GFP-mSK1 to filopodia, suggesting that SK1 regulates different cell biological processes dependent on dimerization. GFP-mSK1 mutants designed to modulate dimerization confirmed this difference in localization. Regulation by the C-terminal tail of SK1 was investigated using GFP-mSK1 truncations. Removal of the last five amino acids (PPEEP) prevented translocation of the enzyme to the PM, whereas removal of the last ten amino acids restored translocation. This suggests that the penultimate five amino acids (SRRGP) function as a translocation brake, which can be released by sequestration of the PPEEP sequence. We propose that these determinants alter the arrangement of N-terminal and C-terminal domains in SK1, leading to unique surfaces that promote differential translocation to the PM., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The sphingosine kinases, SK1 and SK2, catalyse the formation of the bioactive signalling lipid, sphingosine 1-phosphate (S1P), from sphingosine. SK1 and SK2 differ in their subcellular localisation, trafficking and regulation, but the isoforms are also distinct in their selectivity toward naturally occurring and synthetic ligands as substrates and inhibitors. To date, only the structure of SK1 has been determined, and a structural basis for selectivity differences in substrate handling by SK2 has yet to be established. Here we present a structural rationale, based on homology modelling and ligand docking, to account for the capacity of SK2, but not SK1, to efficiently process the pharmacologically active substances, fingolimod (FTY720) and safingol, as substrates. We propose that two key residue differences in hSK2 (Ser305/Thr584 in place of Ala175/Ala339 in hSK1) facilitate conformational switching in the lipid head group anchor residue, Asp308 (corresponding to Asp178 in hSK1), to accommodate substrate diversity for SK2. Our analysis accounts for the contrasting behaviour of fingolimod and safingol as non-turnover inhibitors of SK1, but substrates for SK2, and the observed stereoselectivity for phosphorylation of the pro-S hydroxymethyl group of fingolimod to generate (S)-FTY720-P in vivo. We also rationalise why methylation of the pro-R hydroxymethyl of FTY720 switches the behaviour of the resulting compound, (R)-FTY720 methyl ether (ROMe), to SK2-selective inhibition. Whilst the pharmacological significance of (S)-FTY720-P is firmly established, as the active principle of fingolimod in treating relapsing-remitting multiple sclerosis, the potential importance of SK-mediated phosphorylation of other substrates, such as safingol and non-canonical naturally occuring substrates such as (4E,nZ)-sphingadienes, is less widely appreciated. Thus, the contribution of SK2-derived safingol 1-phosphate to the anti-cancer activity of safingol should be considered. Similarly, the biological role of sphingadiene 1-phosphates derived from plant-based dietary sphingadienes, which we also show here are substrates for both SK1 and SK2, merits investigation., (Copyright © 2020. Published by Elsevier Inc.)

Sphingosine kinases (SK) catalyze the phosphorylation of sphingosine to generate sphingosine-1-phosphate. Two isoforms of SK (SK1 and SK2) exist in mammals. Previously, we showed the beneficial effects of SK2 inhibition, using ABC294640, in a psoriasis mouse model. However, ABC294640 also induces the degradation of SK1 and dihydroceramide desaturase 1 (DES1). Considering these additional effects of ABC294640, we re-examined the efficacy of SK2 inhibition in an IMQ-induced psoriasis mouse model using a novel SK2 inhibitor, HWG-35D, which exhibits nM potency and 100-fold selectivity for SK2 over SK1. Topical application of HWG-35D ameliorated IMQ-induced skin lesions and normalized the serum interleukin-17A levels elevated by IMQ. Application of HWG-35D also decreased skin mRNA levels of interleukin-17A, K6 and K16 genes induced by IMQ. Consistent with the previous data using ABC294640, HWG-35D also blocked T helper type 17 differentiation of naïve CD4 + T cells with concomitant reduction of SOCS1. Importantly, HWG-35D did not affect SK1 or DES1 expression levels. These results reaffirm an important role of SK2 in the T helper type 17 response and suggest that highly selective and potent SK2 inhibitors such as HWG-35D might be of therapeutic use for the treatment of psoriasis.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to a family of G protein-coupled receptors (S1P 1-5 ) and intracellular targets, such as HDAC1/2, that are functional in normal and pathophysiologic cell biology. There is a significant role for sphingosine 1-phosphate in cancer underpinning the so-called hallmarks, such as transformation and replicative immortality. In this review, we survey the most recent developments concerning the role of sphingosine 1-phosphate receptors, sphingosine kinase and S1P lyase in cancer and the prognostic indications of these receptors and enzymes in terms of disease-specific survival and recurrence. We also provide evidence for identification of new therapeutic approaches targeting sphingosine 1-phosphate to prevent neovascularisation, to revert aggressive and drug-resistant cancers to more amenable forms sensitive to chemotherapy, and to induce cytotoxicity in cancer cells. Finally, we briefly describe current advances in the development of isoform-specific inhibitors of sphingosine kinases for potential use in the treatment of various cancers, where these enzymes have a predominant role. This review will therefore highlight sphingosine 1-phosphate signalling as a promising translational target for precision medicine in stratified cancer patients., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cancer stem cells (CSCs) represent rare tumor cell populations capable of self-renewal, differentiation, and tumor initiation and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches that can effectively target CSCs and tumor cells could be the key to efficient tumor treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibited cell proliferation and induced apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhanced breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signaling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrated that SPHK1 inhibitors, FTY720 and PF543, synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies., Competing Interests: The authors declare no conflict of interest.

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells., Competing Interests: Declaration of competing interest All authors declare no conflict of interest regarding the manuscript entitled ‘The regulation of p53, p38 MAPK, JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells’., (Copyright © 2020. Published by Elsevier B.V.)

There is substantial evidence that the enzymes, sphingosine kinase 1 and 2, which catalyse the formation of the bioactive lipid sphingosine 1-phosphate, are involved in pathophysiological processes. In this chapter, we appraise the evidence that both enzymes are druggable and describe how isoform-specific inhibitors can be developed based on the plasticity of the sphingosine-binding site. This is contextualised with the effect of sphingosine kinase inhibitors in cancer, pulmonary hypertension, neurodegeneration, inflammation and sickling.

Sphingosine kinase [(SK), isoforms SK1 and SK2] catalyzes the formation of the bioactive lipid, sphingosine 1-phosphate (S1P). This can be exported from cells and bind to S1P receptors to modulate vascular function. We investigated the effect of short-term hypoxia on SK1 expression and the response of arteries to S1P. SK1 expression in rat aortic and coronary artery endothelial cells was studied using immunofluorescence and confocal microscopy. Responses of rat aortic rings were studied using wire myography and reversible hypoxia induced by bubbling myography chambers with 95% N 2 :5% CO 2 Inhibitors were added 30 minutes before induction of hypoxia. S1P induced endothelium-dependent vasodilation via activation of S1P 3 receptors and generation of nitric oxide. Hypoxia significantly increased relaxation to S1P and this was attenuated by (2 R )-1-[[(4-[[3-methyl-5-[(phenylsulfonyl)methyl] phenoxy]methyl]phenyl]methyl]-2-pyrrolidinemethanol [(PF-543), SK1 inhibitor] but not ( R )-FTY720 methyl ether [(ROMe), SK2 inhibitor]. Hypoxia also increased vessel contractility to the thromboxane mimetic, 9,11-dideoxy-11 α ,9 α -epoxymethanoprostaglandin F2 α , which was further increased by PF-543 and ROMe. Hypoxia upregulated SK1 expression in aortic and coronary artery endothelial cells and this was blocked by PF-543 and 2-( p -hydroxyanilino)-4-( p -chlorophenyl)thiazole [(SKi), SK1/2 inhibitor]. The effects of PF-543 and SKi were associated with increased proteasomal/lysosomal degradation of SK1. A short period of hypoxia increases the expression of SK1, which may generate S1P to oppose vessel contraction. Under hypoxic conditions, upregulation of SK1 is likely to lead to increased export of S1P from the cell and vasodilation via activation of endothelial S1P 3 receptors. These data have significance for perfusion of tissue during episodes of ischemia., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Cyclic AMP (cAMP) phosphodiesterase-4 (PDE4) enzymes degrade cAMP and underpin the compartmentalization of cAMP signaling through their targeting to particular protein complexes and intracellular locales. We describe the discovery and characterization of a small-molecule compound that allosterically activates PDE4 long isoforms. This PDE4-specific activator displays reversible, noncompetitive kinetics of activation (increased V max with unchanged K m ), phenocopies the ability of protein kinase A (PKA) to activate PDE4 long isoforms endogenously, and requires a dimeric enzyme assembly, as adopted by long, but not by short (monomeric), PDE4 isoforms. Abnormally elevated levels of cAMP provide a critical driver of the underpinning molecular pathology of autosomal dominant polycystic kidney disease (ADPKD) by promoting cyst formation that, ultimately, culminates in renal failure. Using both animal and human cell models of ADPKD, including ADPKD patient-derived primary cell cultures, we demonstrate that treatment with the prototypical PDE4 activator compound lowers intracellular cAMP levels, restrains cAMP-mediated signaling events, and profoundly inhibits cyst formation. PDE4 activator compounds thus have potential as therapeutics for treating disease driven by elevated cAMP signaling as well as providing a tool for evaluating the action of long PDE4 isoforms in regulating cAMP-mediated cellular processes., Competing Interests: Conflict of interest statement: D.J.P.H., F.O., J.E.F., R.W.A., Z.J., G.C., C.M., A.L.M., K.B., J.M.A., D.R.A., and M.D.H. are employees of Mironid, Limited. N.J.P., E.K., and A.C.M.O. act as consultants to Mironid, Limited., (Copyright © 2019 the Author(s). Published by PNAS.)

Sphingosine kinase enzymes (SK1 and SK2) catalyze the conversion of sphingosine into sphingosine 1-phosphate and play a key role in lipid signaling and cellular responses. Mapping of isoform amino acid sequence differences for SK2 onto the recently available crystal structures of SK1 suggests that subtle structural differences exist in the foot of the lipid-binding "J-channel" in SK2, the structure of which has yet to be defined by structural biology techniques. We have probed these isoform differences with a ligand series derived from the potent SK1-selective inhibitor, PF-543. Here we show how it is possible, even with relatively conservative changes in compound structure, to systematically tune the activity profile of a ligand from ca. 100-fold SK1-selective inhibition, through equipotent SK1/SK2 inhibition, to reversed 100-fold SK2 selectivity, with retention of nanomolar potency.

The increased adipose tissue mass of obese individuals enhances the risk of metabolic syndrome, type 2 diabetes and cardiovascular diseases. During pathological expansion of adipose tissue, multiple molecular controls of lipid storage, adipocyte turn-over and endocrine secretion are perturbed and abnormal lipid metabolism results in a distinct lipid profile. There is a role for ceramides and sphingosine 1-phosphate (S1P) in inducing adipose dysfunction. For instance, the alteration of ceramide biosynthesis, through the de-regulation of key enzymes, results in aberrant formation of ceramides (e.g. C 16:0 and C 18:0 ) which block insulin signaling and promote adipose inflammation. Furthermore, S1P can induce defective adipose tissue phenotypes by promoting chronic inflammation and inhibiting adipogenesis. These abnormal changes are discussed in the context of possible therapeutic approaches to re-establish normal adipose function and to, thereby, increase insulin sensitivity in type 2 diabetes. Such novel approaches include blockade of ceramide biosynthesis using inhibitors of sphingomyelinase or dihydroceramide desaturase and by antagonism of S1P receptors, such as S1P 2 ., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P 1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P 1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P 1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival, with studies showing that it can both promote and protect against apoptosis. We have therefore investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor SKi [2-( p -hydroxyanilino)-4-( p -chlorophenyl)thiazole] or fenretinide, but not the Degs1 inhibitor GT11 { N -[(1 R ,2 S )-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octan-amide}, induced the polyubiquitination of Degs1 ( M r = 40 to 140 kDa) via a mechanism involving oxidative stress, p38 mitogen-activated protein kinase (MAPK), and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit "gain of function" and activate prosurvival pathways, p38 MAPK, c-Jun N-terminal kinase (JNK), and X-box protein 1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], at concentrations of 25 to 50 μM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast to SKi, ABC294640 (25 μM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from proapoptotic to prosurvival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme in cell survival/apoptosis., (Copyright © 2018 American Society for Microbiology.)

We demonstrate here that the G protein-coupled receptor (GPCR), sphingosine 1-phosphate receptor 2 (S1P 2 , Mr = 40 kDa) is shed in hsp70 + and CD63 + containing exosomes from MDA-MB-231 breast cancer cells. The receptor is taken up by fibroblasts, where it is N-terminally processed to a shorter form (Mr = 36 kDa) that appears to be constitutively active and able to stimulate the extracellular signal regulated kinase-1/2 (ERK-1/2) pathway and DNA synthesis. An N-terminally truncated construct of S1P 2 , which may correspond to the processed form of the receptor generated in fibroblasts, was found to be constitutively active when over-expressed in HEK293 cells. Analysis based on the available crystal structure of the homologous S1P 1 receptor suggests that, in the inactive-state, the N-terminus of S1P 2 may tension TM1 so as to maintain a compressive action on TM7. This in turn may stabilise a closed basal state interface between the intracellular ends of TM7 and TM6. Cleavage and removal of the S1P 2 N-terminal peptide is postulated to facilitate relaxation of TM1 and accompanying separation of TM6 and TM7. The latter transition is one of the key elements of G protein engagement and is required to open the intracellular coupling interface beneath the GPCR helix bundle. Therefore, removal at the N-terminus of S1P 2 is likely to enhance G protein coupling. These findings provide the first evidence that S1P 2 is released from breast cancer cells in exosomes and is processed by fibroblasts to promote ERK signaling and proliferation of these cells., Competing Interests: CONFLICTS OF INTEREST The authors report no conflicts of interest in this work.

The bioactive lipid, sphingosine 1-phosphate (S1P) is produced by phosphorylation of sphingosine and this is catalysed by two sphingosine kinase isoforms (SK1 and SK2). Here we discuss structural functional aspects of SK1 (which is a dimeric quaternary enzyme) that relate to coordinated coupling of membrane association with phosphorylation of Ser225 in the 'so-called' R-loop, catalytic activity and protein-protein interactions (e.g. TRAF2, PP2A and G q ). S1P formed by SK1 at the plasma-membrane is released from cells via S1P transporters to act on S1P receptors to promote tumorigenesis. We discuss here an additional novel mechanism that can operate between cancer cells and fibroblasts and which involves the release of the S1P receptor, S1P 2 in exosomes from breast cancer cells that regulates ERK-1/2 signalling in fibroblasts. This novel mechanism of signalling might provide an explanation for the role of S1P 2 in promoting metastasis of cancer cells and which is dependent on the micro-environmental niche., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sphingosine kinase 1 (SphK1) knockout mice are protected against pulmonary hypertension and expression levels of the enzyme are increased in the lungs of pulmonary arterial hypertensive (PAH) patients. Moreover, sphingosine 1-phosphate can promote vascular remodeling/vasoconstriction in rodent and human pulmonary arterial smooth muscle cell models. Therefore, SphK1 might be a novel target for treatment of PAH. However, in our opinion, more refined strategies to target SphK1 are needed because this enzyme is protective against endothelial dysfunction and can become resistant to SphK1 inhibitors in vascular smooth muscle, thereby potentially limiting their effectiveness in PAH. In addition, SphK1 is involved in maladaptive hypertrophy and we propose that heart failure might be an additional direct target for therapeutic intervention with SphK1 inhibitors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The purpose of this Opinion is to present a case for targeting sphingosine kinase 2 (SK2) in autoimmune/inflammatory disease. Data obtained using Sphk2 -/- mice suggest that SK2 is an anti-inflammatory enzyme, although this might be misleading because of a compensatory increase in the expression of a second isoform, sphingosine kinase 1 (SK1), which functions as a proinflammatory enzyme. SK2 is involved in regulating interleukin (IL)-12/interferon gamma (IFN-γ) and histone deacetylase-1/2 (HDAC-1/2) signalling and, potentially, retinoid-related orphan receptor gamma t (ROR-γt) stability linked with T helper (Th) 17 cell polarisation. Therefore, there is a need to develop highly potent SK2 inhibitors with selectivity over SK1 to clearly define the role of SK2 in autoimmune/inflammatory disease. Structural determinants of SK2 relative to SK1 will enable the design of selective SK2 inhibitors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The bioactive lipid, sphingosine 1-phosphate (S1P) binds to a family of G protein-coupled receptors, termed S1P₁-S1P₅. These receptors function in, for example, the cardiovascular system to regulate vascular barrier integrity and tone, the nervous system to regulate neuronal differentiation, myelination and oligodendrocyte/glial cell survival and the immune system to regulate T- and B-cell subsets and trafficking. S1P receptors also participate in the pathophysiology of autoimmunity, inflammatory disease, cancer, neurodegeneration and others. In this review, we describe how S1P₁ can form a complex with G-protein and β-arrestin, which function together to regulate effector pathways. We also discuss the role of the S1P₁-Platelet derived growth factor receptor β functional complex (which deploys G-protein/β-arrestin and receptor tyrosine kinase signaling) in regulating cell migration. Possible mechanisms by which different S1P-chaperones, such as Apolipoprotein M-High-Density Lipoprotein induce biological programmes in cells are also described. Finally, the role of S1P₁ in health and disease and as a target for clinical intervention is appraised.

Background and Purpose: The sphingosine analogue, FTY720 (Gilenya R ), alleviates clinical disease progression in multiple sclerosis. Here, we variously assessed the effects of an azide analogue of (S)-FTY720 vinylphosphonate (compound 5; a sphingosine kinase 1 activator), (R)-FTY720 methyl ether (ROMe, a sphingosine kinase 2 inhibitor) and RB-020 (a sphingosine kinase 1 inhibitor and sphingosine kinase 2 substrate) on IL-1β formation, sphingosine 1-phosphate levels and expression of S1P 1 receptors. We also assessed the effect of compound 5 and ROMe in an experimental autoimmune encephalomyelitis (EAE) model in mice., Experimental Approach: We measured IL-1β formation by macrophages, sphingosine 1-phosphate levels and expression levels of S1P 1 receptors in vitro and clinical score in mice with EAE and the extent of inflammatory cell infiltration into the spinal cord in vivo., Key Results: Treatment of differentiated U937 macrophages with compound 5, RB-020 or sphingosine (but not ROMe) enhanced IL-1β release. These data suggest that these compounds might be pro-inflammatory in vitro. However, compound 5 or ROMe reduced disease progression and infiltration of inflammatory cells into the spinal cord in EAE, and ROMe induced a reduction in CD4 + and CD8 + T-cell levels in the blood (lymphopenia). Indeed, ROMe induced a marked decrease in expression of cell surface S1P 1 receptors in vitro., Conclusion and Implications: This is the first demonstration that an activator of sphingosine kinase 1 (compound 5) and an inhibitor of sphingosine kinase 2 (ROMe, which also reduces cell surface S1P 1 receptor expression) have an anti-inflammatory action in EAE., (© 2016 The British Pharmacological Society.)

Sphingolipids, such as ceramide, sphingosine and sphingosine 1-phosphate (S1P) are bioactive molecules that have important functions in a variety of cellular processes, which include proliferation, survival, differentiation and cellular responses to stress. Sphingolipids have a major impact on the determination of cell fate by contributing to either cell survival or death. Although ceramide and sphingosine are usually considered to induce cell death, S1P promotes survival of cells. Sphingosine kinases (SPHKs) are the enzymes that catalyze the conversion of sphingosine to S1P. There are two isoforms, SPHK1 and SPHK2, which are encoded by different genes. SPHK1 has recently been implicated in contributing to cell transformation, tumor angiogenesis and metastatic spread, as well as cancer cell multidrug-resistance. More recent findings suggest that SPHK2 also has a role in cancer progression. This review is an overview of our understanding of the role of SPHKs and S1P in hematopoietic malignancies and provides information on the current status of SPHK inhibitors with respect to their therapeutic potential in the treatment of hematological cancers.

Recent studies have demonstrated that the expression of sphingosine kinase 1, the enzyme that catalyses formation of the bioactive lipid, sphingosine 1-phosphate, is increased in lungs from patients with pulmonary arterial hypertension. In addition, Sk1(-/-) mice are protected from hypoxic-induced pulmonary arterial hypertension. Therefore, we assessed the effect of the sphingosine kinase 1 selective inhibitor, PF-543 and a sphingosine kinase 1/ceramide synthase inhibitor, RB-005 on pulmonary and cardiac remodelling in a mouse hypoxic model of pulmonary arterial hypertension. Administration of the potent sphingosine kinase 1 inhibitor, PF-543 in a mouse hypoxic model of pulmonary hypertension had no effect on vascular remodelling but reduced right ventricular hypertrophy. The latter was associated with a significant reduction in cardiomyocyte death. The protection involves a reduction in the expression of p53 (that promotes cardiomyocyte death) and an increase in the expression of anti-oxidant nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). In contrast, RB-005 lacked effects on right ventricular hypertrophy, suggesting that sphingosine kinase 1 inhibition might be nullified by concurrent inhibition of ceramide synthase. Therefore, our findings with PF-543 suggest an important role for sphingosine kinase 1 in the development of hypertrophy in pulmonary arterial hypertension., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingosine kinases (SK1 and SK2) catalyse the conversion of sphingosine into sphingosine 1-phosphate and control fundamental cellular processes, including cell survival, proliferation, differentiation, migration, and immune function. In this review, we highlight recent breakthroughs in the structural and functional characterisation of SK1 and these are contextualised by analysis of crystal structures for closely related prokaryotic lipid kinases. We identify a putative dimerisation interface and propose novel regulatory mechanisms governing structural plasticity induced by phosphorylation and interaction with phospholipids and proteins. Our analysis suggests that the catalytic function and regulation of the enzymes might be dependent on conformational mobility and it provides a roadmap for future interrogation of SK1 function and its role in physiology and disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sphingosine kinases (isoforms SK1 and SK2) catalyse the formation of a bioactive lipid, sphingosine 1-phosphate (S1P). S1P is a well-established ligand of a family of five S1P-specific G protein coupled receptors but also has intracellular signalling roles. There is substantial evidence to support a role for sphingosine kinases and S1P in health and disease. This review summarises recent advances in the area in relation to receptor-mediated signalling by S1P and novel intracellular targets of this lipid. New evidence for a role of each sphingosine kinase isoform in cancer, the cardiovascular system, central nervous system, inflammation and diabetes is discussed. There is continued research to develop isoform selective SK inhibitors, summarised here. Analysis of the crystal structure of SK1 with the SK1-selective inhibitor, PF-543, is used to identify residues that could be exploited to improve selectivity in SK inhibitor development for future therapeutic application., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

We have assessed the effect of two ether glycerol lipids, 77-6 ((2S, 3R)-4-(Tetradecyloxy)-2-amino-1,3-butanediol) and 56-5 ((S)-2-Amino-3-O-hexadecyl-1-propanol), which are substrates for sphingosine kinases, on inflammatory responses. Treatment of differentiated U937 macrophage-like cells with 77-6 but not 56-5 enhanced IL-1β release; either alone or in the presence of LPS. The stimulatory effect of sphingosine or 77-6 on LPS-stimulated IL-1β release was reduced by pretreatment of cells with the caspase-1 inhibitor, Ac-YVAD-CHO, thereby indicating a role for the inflammasome. The enhancement of LPS-stimulated IL-1β release in response to sphingosine, but not 77-6, was reduced by pretreatment of cells with the cathepsin B inhibitor, CA074Me, indicating a role for lysosomal destabilization in the effect of sphingosine. Administration of 56-5 to mice increased disease progression in an experimental autoimmune encephalomyelitis model and this was associated with a considerable increase in the infiltration of CD4(+) T-cells, CD11b(+) monocytes and F4/80(+) macrophages in the spinal cord. 56-5 and 77-6 were without effect on the degradation of myc-tagged sphingosine 1-phosphate 1 receptor in CCL39 cells. Therefore, the effect of 56-5 on EAE disease progression is likely to be independent of the inflammasome or the sphingosine 1-phosphate 1 receptor. However, 56-5 is chemically similar to platelet activating factor and the exacerbation of EAE disease progression might be linked to platelet activating factor receptor signaling., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

With the passing of Dr. Robert Bittman from pancreatic cancer on the 1st October 2014, the lipid research field lost one of the most influential and significant personalities. Robert Bittman's genius was in chemical design and his contribution to the lipid research field was truly immense. The reagents and chemicals he designed and synthesised allowed interrogation of the role of lipids in constituting complex biophysical membranes, sterol transfer and in cellular communication networks. Here we provide a review of these works which serve as a lasting memory to his life., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sphingosine kinase (there are two isoforms, SK1 and SK2) catalyses the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that can be released from cells to activate a family of G protein-coupled receptors, termed S1P1-5. In addition, S1P can bind to intracellular target proteins, such as HDAC1/2, to induce cell responses. There is increasing evidence of a role for S1P receptors (e.g. S1P4) and SK1 in cancer, where high expression of these proteins in ER negative breast cancer patient tumours is linked with poor prognosis. Indeed, evidence will be presented here to demonstrate that S1P4 is functionally linked with SK1 and the oncogene HER2 (ErbB2) to regulate mitogen-activated protein kinase pathways and growth of breast cancer cells. Although much emphasis is placed on SK1 in terms of involvement in oncogenesis, evidence will also be presented for a role of SK2 in both T-cell and B-cell acute lymphoblastic leukemia. In patient T-ALL lymphoblasts and T-ALL cell lines, we have demonstrated that SK2 inhibitors promote T-ALL cell death via autophagy and induce suppression of c-myc and PI3K/AKT pathways. We will also present evidence demonstrating that certain SK inhibitors promote oxidative stress and protein turnover via proteasomal degradative pathways linked with induction of p53-and p21-induced growth arrest. In addition, the SK1 inhibitor, PF-543 exacerbates disease progression in an experimental autoimmune encephalomyelitis mouse model indicating that SK1 functions in an anti-inflammatory manner. Indeed, sphingosine, which accumulates upon inhibition of SK1 activity, and sphingosine-like compounds promote activation of the inflammasome, which is linked with multiple sclerosis, to stimulate formation of the pro-inflammatory mediator, IL-1β. Such compounds could be exploited to produce antagonists that diminish exaggerated inflammation in disease. The therapeutic potential of modifying the SK-S1P receptor pathway in cancer and inflammation will therefore, be reviewed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Resveratrol, a natural compound endowed with multiple health-promoting effects, has received much attention given its potential for the treatment of cardiovascular, inflammatory, neurodegenerative, metabolic and age-related diseases. However, the translational potential of resveratrol has been limited by its specificity, poor bioavailability and uncertain toxicity. In recent years, there has been an accumulation of evidence demonstrating that resveratrol modulates sphingolipid metabolism. Moreover, resveratrol forms higher order oligomers that exhibit better selectivity and potency in modulating sphingolipid metabolism. This review evaluates the evidence supporting the modulation of sphingolipid metabolism and signaling as a mechanism of action underlying the therapeutic efficacy of resveratrol and oligomers in diseases, such as cancer.

The most potent inhibitor of Sphingosine Kinase 1 (SPHK1) so far identified is PF-543. The crystal structure of SPHK1 in complex with inhibitor PF-543 to 1.8 Å resolution reveals the inhibitor bound in a bent conformation analogous to that expected of a bound sphingosine substrate but with a rotated head group. The structural data presented will aid in the design of SPHK1 and SPHK2 inhibitors with improved properties.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.

In this issue of Oncogene, Albinet et al. have demonstrated a critical role of melanoma sphingosine kinase 1, which catalyses formation of sphingosine 1-phosphate (S1P), in promoting the differentiation of fibroblasts into myofibroblasts. The myofibroblast sphingosine kinase 1 then promotes the S1P-dependent dissemination (metastasis) of melanoma cells via a S1P receptor 3-mediated mechanism. These findings are of major significance because they provide a novel mechanism of interaction between melanoma and the microenvironment niche in promoting metastasis. These studies therefore identify S1P derived from myofibroblasts and melanoma cells as a novel target for therapeutic intervention.

We demonstrate that pre-treatment of estrogen receptor negative MDA-MB-231 breast cancer cells containing ectopically expressed HA-tagged sphingosine 1-phosphate receptor-2 (S1P2) with the sphingosine kinase 1/2 inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) or the sphingosine kinase 2 selective inhibitor (R)-FTY720 methyl ether (ROMe) or sphingosine kinase 2 siRNA induced the translocation of HA-tagged S1P2 and Y416 phosphorylated c-Src to the nucleus of these cells. This is associated with reduced growth of HA-tagged S1P2 over-expressing MDA-MB-231 cells. Treatment of HA-S1P2 over-expressing MDA-MB-231 cells with the sphingosine 1-phosphate receptor-4 (S1P4) antagonist CYM50367 or with S1P4 siRNA also promoted nuclear translocation of HA-tagged S1P2. These findings identify for the first time a signaling pathway in which sphingosine 1-phosphate formed by sphingosine kinase 2 binds to S1P4 to prevent nuclear translocation of S1P2 and thereby promote the growth of estrogen receptor negative breast cancer cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)