Investigation of sphingosine kinase 1 inhibitory potential of cinchonine and colcemid targeting anticancer therapy

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.

Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

Sphingosine 1-phosphate (S1P) is an important regulator of many different immune functions including lymphocyte circulation, antigen presentation, and T cell development. It stimulates five G protein-coupled receptors designated S1P_1-5, which are also expressed by immune cells. S1P receptors couple to different heterotrimeric G proteins including G alpha i, q, and 12/13, and elicit cellular signalling events by activating the small GTPases Rac and Rho and protein kinases Akt, ERK, and JNK, and by inducing cellular calcium flux and inhibiting cAMP accumulation, amongst others. S1P is the exit signal for lymphocytes leaving lymphoid organs and present in blood and lymph at high nanomolar concentrations due to the S1P-producing activity of sphingosine kinases (SK). The S1P-degrading enzyme S1P-lyase maintains low amounts of S1P in lymphoid organs. Disrupting this concentration difference by S1P receptor agonists and antagonists like FTY720, SEW2871, and VPC23019, by an anti-S1P antibody, or by inhibiting the S1P-lyase has therapeutic potential for autoimmune diseases like multiple sclerosis (MS) and rheumatoid arthritis and for many other disorders like cancer, fibrosis, inflammation, macular degeneration, diabetic retinopathy, and glaucoma. This report aims to provide a brief overview of concepts, approaches, pharmaceutical compounds, and targets that are currently used to modulate S1P-driven immune functions.

Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p< 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p<0.01 after 3 days). SK activity could be induced further in SKAP cells by the PKC activator PMA, although to a lesser extent than in vector alone cells, while the addition of MK-571 and FTC resulted in a marked increase in intracellular S1P in SKAP and vector alone cells, with a decrease in extracellular S1P levels. These experiments confirm the membrane localisation of SK1 in SKAP transfected cells. SKAP transfection did not affect sensitivity to imatinib or doxorubicin compared to vector alone. Conclusion: These data suggest that SKAP may act as a positive regulator of SK1 activity in cancer cells, an observation that has implications in carcinogenesis and chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256

Sphingosine-1-phosphate (S1P), via interaction with its G protein-coupled receptors, regulates various physiological and pathological responses. The present study investigated the role of S1P/S1P receptor signaling in several functional responses of human fibroblast-like synoviocytes (FLSs) that may contribute to the pathogenesis of rheumatoid arthritis (RA). We report that FLSs express the S1P(1), S1P(2), and S1P(3) receptors. Moreover, exogenously applied S1P induces FLS 1) migration, 2) secretion of inflammatory cytokines/chemokines, and 3) protection from apoptosis. Using specific S1P receptor agonists/antagonists, we determined that S1P stimulates FLS migration through S1P(1) and S1P(3), induces cytokine/chemokine secretion through S1P(2) and S1P(3), and protects from cell apoptosis via S1P(1). The S1P-mediated cell motility and cytokine/chemokine secretion seem to be regulated by the p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and Rho kinase signal transduction pathways. Interestingly, treatment of FLSs with tumor necrosis factor-alpha increases S1P(3) expression and correlates with the enhancement of S1P-induced cytokine/chemokine production. Our data suggest that S1P(1), S1P(2), and S1P(3) play essential roles in the pathogenesis of RA by modulating FLS migration, cytokine/chemokine production, and cell survival. Moreover, the cytokine-rich environment of the inflamed synovium may synergize with S1P signaling to exacerbate the clinical manifestations of this autoimmune disease.

Background: Sphingosine kinase 1 (SPHK1) is over-expressed in multiple cancers including breast and colon cancer. SPHK1 catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). S1P promotes tumorigenesis by inhibiting apoptosis and increasing cell proliferation and angiogenesis. Thus SPHK1 has been evaluated as a therapeutic target in cancer. The antidiabetic drug, metformin, has been reported to have protective effects in several cancers, including ovarian cancer. However, molecular mechanisms mediating the anti-tumor effects of metformin are not fully understood. In this study, we demonstrate that SPHK1 is a novel target of metformin and may predict metformin response in ovarian cancer. Methods: The S1P signaling cascade was profiled in several ovarian cancer cell lines. Four different ovarian cancer cell lines were treated with 1mM and 5mM metformin and the mRNA and protein levels of SPHK1 were measured by qRT-PCR and western blotting respectively. Next, the Tyk-nu, HeyA8, SNU119, Kuramochi cell lines were stably transfected to overexpression SPHK1 and metformin sensitivity was measured using MTT proliferation assays. Metabolomic analysis was performed on serum from ovarian cancer patients using metformin for diabetes and compared controls (IRB 13248A). Serum samples were analyzed using a Q-Exactive orbitrap mass spectrometer coupled to a Dionex ultimate UHPLC. Results: In all cell lines tested, metformin suppresses SPHK1 mRNA and protein levels. Analysis of the S1P signaling pathway showed that metformin sensitive cell lines (Tyknu and HeyA8) have high SPHK1 and low S1P lyase (SGPL1) expression, the opposite is true in metformin resistant cell lines (Kuramochi and SNU119). At the same time, S1P receptor 1 (S1PR1) is the main S1P receptor among five S1P receptors (S1PR1-S1PR5) in metformin sensitive cancer cells, however S1PR1 is not dominant in metformin resistant cancer cells. Overexpression of SPHK1 upregulates S1PR1 and increases metformin sensitivity in both metformin sensitive and resistant cell lines. The in vitro findings are augmented by preliminary metabolomic analysis of patient samples that revealed that ovarian cancer patients using metformin for diabetes have lower serum levels of S1P compared to controls. Conclusions: The findings of this study indicate that SPHK1 is a novel metformin target in ovarian cancer. Specifically, high SPHK1 expression sensitizes cells to metformin and S1P signaling profiles predicts metformin response. Based on these findings we propose that sphingolipid signaling be evaluated as a metformin-response signature in ongoing clinical trials of the drug as adjuvant treatment for ovarian cancer (NCT02122185). Citation Format: Tatsuyuki Chiyoda, Xiaojing Liu, Ernst Lengyel, Jason Locasale, Iris Romero. Sphingosine kinase 1 as a mediator and predictor of metformin's protective effect in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A68.

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Investigating potential of cholic acid, syringic acid, and mangiferin as cancer therapeutics through sphingosine kinase 1 inhibition

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K(m)(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.

Sphingosine kinase-1 (SPHK1) is an enzyme that catalyzes the formation of the prosurvival second messenger sphingosine-1-phosphate (S1P) from the proapoptotic lipid sphingosine. The balance between sphingosine and S1P forms a sphingolipid rheostat that is primed for cell death when the balance shifts towards sphingosine/ceramide or to cell survival when S1P levels are increased. Elevated levels of S1P have been observed in several cancers, including ovarian cancer where it is elevated in both the ascites and serum of patients. Correspondingly, high expression of SPHK1 has also been described in multiple cancer types and has been linked to disease progression. Here we report that SPHK1 is overexpressed in a subset of epithelial ovarian cancers (EOC) and correlates with poor progression-free survival. Overexpression and knockdown of SPHK1 in multiple human EOC cell lines modulates in vitro cell proliferation, anchorage-independent growth, and chemosensitivity. In mouse xenograft studies, intraperitoneal administration of a SPHK1-specific inhibitor (SKI-5c) decreases tumor size, indicating that SPHK1 may be a potential therapeutic target in EOC. S1P is a secreted factor that has been shown to influence the tumor microenvironment of other cancers; however its role in EOC has not been fully established. Using a co-culture model, we show that overexpression of SPHK1 in EOC cells stimulates the transition of normal ovarian stromal fibroblasts to myofibroblasts, and enhances stromal SPHK1 expression. In a reciprocal manner, overexpression of SPHK1 in stromal fibroblast results in increased expression of MMP2 and MMP9 in EOC cells, which may contribute to a more invasive phenotype. Interestingly, stromal SPHK1 expression also enhanced expression of SPHK1 and the S1P receptor (S1PR1) in the EOC cells. Biostatistical analysis suggests a significant correlation between SPHK1 expression in EOC cells and the expression of several extracellular matrix genes (FN1, POSTN, VCAN), which have been shown to have role in the EOC tumor microenvironment. Overall, these results suggest that SPHK1 is a critical regulator of ovarian tumor cell proliferation and survival, and a mediator of tumor-stroma interaction. Citation Format: Jessica A. Beach, Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel C. Gozo, Beth Y. Karlan, Sandra Orsulic. Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B09. doi:10.1158/1538-7445.CHTME14-B09

Sphingosine kinase 1 (SphK1) is an essential enzyme in sphingolipid metabolism, catalyzing the phosphorylation of sphingosine to produce sphingosine-1-phosphate (S1P), a bioactive lipid with diverse roles in cell proliferation, survival, and migration. Dysregulation of the SphK1/S1P axis is implicated in a variety of pathological conditions, including inflammatory, metabolic, and neurodegenerative diseases. Targeting SphK1 represents a promising therapeutic strategy, particularly in oncology and inflammation-related pathologies. In this study, we investigated the potential of three natural compounds, Baicalin (BA), Naringenin (NR), and Noscapine (NS) as SphK1 inhibitors. Through combined molecular docking, molecular dynamics simulations, binding studies and enzyme inhibition assays, we identified these compounds as effective SphK1 inhibitors. BA, NR, and NS exhibited binding affinities characterized by IC50 values of 26.542, 32.157, and 28.134μM, respectively. These molecules bind to the active site of SphK1 with favorable binding energies with strong non-covalent interactions. This study provides structural and functional insights into potential of BA, NR, and NS to target SphK1 selectively, which can function as lead compounds for developing novel anti-cancer therapy with minimal off-target effects, offering avenues for developing drugs with enhanced specificity and affinity for this enzyme.

Sphingosine 1-phosphate (S1P), a multi-functional phospholipid mediator, is generated from sphingosine by sphingosine kinases and binds to five known G protein-coupled S1P receptors (S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅). It is widely accepted that S1P receptor 1 (S1P₁) plays an essential role in lymphocyte egress from the secondary lymphoid organs (SLO) and thymus, because lymphocyte egress from these organs to periphery is at extremely low levels in mice lacking lymphocytic S1P₁. Fingolimod hydrochloride (FTY720) is a first-in-class, orally active S1P₁ functional antagonist which was discovered by chemical modification of a natural product, myriocin. Since FTY720 has a structure closely related to sphingosine, the phosphorylated FTY720 (FTY720-P) is converted by sphingosine kinases and binds 4 types of S1P receptors. FTY720-P strongly induces down-regulation of S1P₁ by internalization and degradation of this receptor and acts as a functional antagonist at S1P₁. Consequently, FTY720 inhibits S1P₁-dependent lymphocyte egress from the SLO and thymus to reduce circulating lymphocytes including autoreactive Th17 cells, and is highly effective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In relapsing remitting MS patients, oral FTY720 shows a superior efficacy when compared to intramuscular interferon-β-1a. Based on these data, it is presumed that modulation of the S1P-S1P₁ axis provides an effective therapy for autoimmune diseases including MS.