Targeting SphK1 Research Articles

Inhibition of sphingosine kinase 1 attenuates LPS-induced acute lung injury by suppressing endothelial cell pyroptosis

Acute lung injury (ALI) is a frequent complication of sepsis, with pyroptosis playing a pivotal role. Analysis of Gene Expression Omnibus (GEO) mouse sepsis datasets revealed the upregulation of sphingosine kinase 1 (SphK1) in septic mouse lung tissues, which was validated in lipopolysaccharide (LPS)-treated mice. Therefore, this study aimed to explore the potential role and underlying mechanisms of SphK1, the primary kinase responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphat, in sepsis development. Mice received an intraperitoneal injection of SphK1 inhibitor prior to LPS administration. Mouse lung vascular endothelial cells (MLVECs) were exposed to LPS and SphK1 inhibitor. The SphK1 inhibitor mitigated ALI, as evidenced by hematoxylin and eosin (H&E) staining and the wet-to-dry (W/D) weight ratio and reduced Evans blue dye leakage. Furthermore, the SphK1 inhibitor inhibited the activation of the NOD-like receptor protein 3 inflammasome and the subsequent induction of pyroptosis both in vivo and in vitro. Intriguingly, using co-immunoprecipitation (Co-IP) combined with mass spectrometry, our findings revealed that SphK1 associates with pyruvate kinase M2 (PKM2), facilitating PKM2 phosphorylation and its nuclear translocation. TEPP-46, which has the ability to stabilize PKM2 and inhibit the phosphorylation and nuclear translocation of PKM2, markedly reduced the expression of pyroptosis-associated markers and alleviated lung injury. Concludingly, our results suggest that targeting SphK1 is a promising therapeutic strategy for ALI.

174P Targeting SPHK1 in macrophages suppresses liver metastasis of colorectal cancer and decouples anti-tumor immunity from immunotherapy toxicity

174P Targeting SPHK1 in macrophages suppresses liver metastasis of colorectal cancer and decouples anti-tumor immunity from immunotherapy toxicity

Atractylenolide-1 targets SPHK1 and B4GALT2 to regulate intestinal metabolism and flora composition to improve inflammation in mice with colitis

BackgroundAtractylenolide-1, an active component of Atractylodes Lancea, which is widely used to improve the gastrointestinal function. However, the efficacy and mechanism remain unclear in treating ulcerative colitis (UC). PurposeThis study aimed to investigate the efficacy and the underlying mechanism of atractylenolide-1in UC. MethodsA dextran sulfate sodium (DSS)-induced UC mouse model was used to investigate the efficacy of atractylenolide-1. 16S DNA sequencing, GC–MS technique and transcriptome sequencing were used to detect the composition of mouse intestinal flora, the changes of metabolites and gene expression in mouse intestine. Compound-reaction-enzyme-gene network was used to find drug targets. Recombinant plasmid overexpression was used to verify drug targets in DSS mouse models. ResultsThe results showed that Atractylenolide-1 could significantly improve weight loss, diarrhea, blood in the stool, shortening of the colon, the loss of colonic goblet cells, reduction in mucoprotein MUC2, and tight junction proteins (zo-1, occludin) in mice with colitis. It reduced the inflammatory factors TNF-α, IL-6, IL-1β as well. The 16S sequencing showed that Atractylenolide-1 regulated the diversity and abundance of the intestinal flora in mice with colitis, and the analysis of flora enrichment indicated that the regulation of intestinal flora by atractylenolide-1 may be related to the regulation of metabolism. Correlation analysis of metabolomics and transcriptome showed that two genes SPHK1 and B4GALT2 related to the metabolism of fructose and galactose were regulated by atractylenolide-1. Further verification showed that atractylenolide-1 significantly inhibited the aberrance of SPHK1 and B4GALT2 in the colon with colitis. Meanwhile, it inhibited the activation of the PI3K-AKT pathway. SPHK1 and B4GALT2 overexpressing reversed the therapeutic effect of atractylenolide-1 in mice with colitis. ConclusionAtractylenolide-1 is a potential drug for the treatment of colitis by suppressing inflammation via the SPHK1/PI3K/AKT axis and by targeting SPHK1 and B4GAT2 to regulate fructose/galactose-related metabolism, thereby regulating the composition of the intestinal flora.

Long Noncoding RNA MALAT1 Interacts with miR-124-3p to Modulate Osteosarcoma Progression by Targeting SphK1.

Introduction Long noncoding RNAs (lncRNAs) have been implicated in a variety of biological functions, including tumor proliferation, apoptosis, progression, and metastasis. lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is overexpressed in various cancers, as well as osteosarcoma (OS); however, its underlying mechanism in OS is poorly understood. This investigation aims to elucidate the mechanisms of MALAT1 in OS proliferation and migration and to provide theoretical grounding for further targeted therapy in OS. Methods In the present study, we applied qRT-PCR to assess the MALAT1 expression in OS tissues and cell lines. The effects of MALAT1 and miR-124-3p on OS cell proliferation and migration were studied by CCK-8 and scratch assays. Cell cycle and apoptosis were tested using a flow cytometer. The competing relationship between MALAT1 and miR-124-3p was confirmed by dual-luciferase reporter assay. Results MALAT1 was overexpressed in OS cell lines and tissue specimens, and knockdown of MALAT1 significantly inhibited cell proliferation and migration and increased cell apoptosis and the percentage of G0/G1 phase. Furthermore, MALAT1 could directly bind to miR-124-3p and inhibit miR-124-3p expression. Moreover, MALAT1 overexpression significantly relieved the inhibition on OS cell proliferation mediated by miR-124-3p overexpression, which involved the derepression of sphingosine kinase 1 (SphK1). Conclusions We propose that lncRNA MALAT1 interacts with miR-124-3p to modulate OS progression by targeting SphK1. Hence, we identified a novel MALAT1/miR-124-3p/SphK1 signaling pathway in the regulation of OS biological behaviors.

L-ascorbyl 6-palmitate as lead compound targeting SphK1: an in silico and in vitro investigation

Sphingosine kinases (SphKs) are a class of lipid kinases, that have received extensive attention as important rate-limiting enzyme in tumor. Inhibition of the activity of SphK1 can lead to an anticancer effect. Herein, we describe the discovery process and biological characteristics of a new SphK1 inhibitor, ascorbyl palmitate, discovered through computer-aided drug design. Biochemical experiments show that ascorbyl palmitate has a strong inhibitory effect on SphK1, with an IC50 value of 6.4 μM. The MTT experiment showed that ascorbyl palmitate had anti-cancer effects toward the U87, A549, 22RV1, and A375 cell lines. Among them, ascorbyl palmitate has prominent inhibitory activity against the 22RV1 cell line, with an IC50 value of 41.57 μM. To explore the structure–activity relationship, four ascorbyl palmitate derivatives were synthesized and tested for kinase activity. The outstanding effect of ascorbyl palmitate toward SphK1 and its known non-toxicity suggest that ascorbyl palmitate may be a lead compound for the development of effective SphK1 anti-cancer inhibitors.

Abstract 13494: Lncrna Khps1 Regulates Hif-1a-dependent Pulmonary Vascular Remodeling by Targeting Mir-1, Sphk1 and S1pr2

Rationale: Pulmonary vascular remodeling is common in pulmonary arterial hypertension (PAH) and is associated with increased risk of right ventricular failure and death. We have previously reported that sphingosine kinase 1 (Sphk1), sphingosine 1 phosphate receptor 2 (S1PR2), microRNA-1-3p (miR-1-3p) and hypoxia-inducible factor-1α (HIF-1α) are involved in the regulation of pulmonary vascular remodeling and the development of PAH. Recently, it has been reported that the long noncoding RNA (LncRNA) Khps1 is required for Sphk1 expression and cell proliferation. However, whether Khps1 plays a role in PAH is unclear. Here, we tested the hypothesis that Khps1 has an impact on pulmonary vascular remodeling in PAH by regulating Sphk1/S1PR2/HIF-1α signaling pathway. Methods and Results: Using qRT-PCR, our data showed that Khps1 expression levels were up-regulated in pulmonary artery smooth muscle cells (PASMCs) from PAH patients. Increased Khps1 expression levels were also observed in human PASMCs exposed to hypoxia and in lungs obtained from monocrotaline (MCT)-treated rats or hypoxia exposed mice. Luciferase reporter assays showed that Khps1 expression is dependent on E2F1-mediated promoter activation. In silico analysis and luciferase reporter assays also suggested that Khps1 can directly bind to miR-1-3p, suggesting that Khps1 functions as a competing endogenous RNA. Similar studies also showed that miR-1-3p regulates expression of Sphk1 and S1PR2 by binding to their 3’UTR regions. Concurrent E2F1 overexpression and khps1 silencing resulted in a decrease in Sphk1 and S1PR2 expression levels, reduced HIF-1α stabilization and suppressed PASMCs proliferation. Finally, over expression of Sphk1 increased HIF-1α and that in turn increased E2F1 expression in human PASMCs. Conclusion: These data reveal a novel regulatory loop between E2F1, Khps1, miR-1-3p and Sphk1/S1PR2 in the regulation of PASMC proliferation and pulmonary vascular remodeling in PAH. These data also suggest Khps1 regulates pulmonary vascular remodeling in a HIF-1α-dependent manner through targeting Sphk1 and S1PR2.

The therapeutic value of the SphK1-targeting microRNA-3677 in human osteosarcoma cells.

Sphingosine kinase 1 (SphK1) is a potential therapeutic target for human osteosarcoma (OS). SphK1-targeting microRNAs (miRNAs) could have important therapeutic value for OS. We discovered that micorRNA-3677 (miR-3677) is a SphK1-targeting miRNA, inhibiting OS cell progression. The results of RNA-Pull down assay confirmed direct binding between biotinylated-miR-3677 and SphK1 mRNA in primary human OS cells. In established and primary human OS cells forced overexpression of miR-3677, by a lentiviral construct, decreased SphK1 3’-UTR (untranslated region) activity and downregulated SphK1 expression. Both were however enhanced with miR-3677 inhibition in OS cells. Function studies demonstrated that OS cell growth, proliferation and migration were inhibited with miR-3677 overexpression, but augmented with miR-3677 inhibition. MiR-3677 overexpression-induced anti-OS cell activity was reversed with re-expression of the 3’-UTR-depleted SphK1. Additionally, in SphK1 knockout OS cells (by CRISPR/Cas9 strategy), altering miR-3677 expression failed to further alter cell functions. Finally, we show that miR-3677 expression was significantly downregulated in primary human OS tissues, correlating with SphK1 mRNA upregulation. We conclude that targeting SphK1 by miR-3677 inhibits human OS cell progression.

Furowanin A Exhibits Antiproliferative and Pro-Apoptotic Activities by Targeting Sphingosine Kinase 1 in Osteosarcoma.

Osteosarcoma (OS) is one of the most common malignant bone tumors among children and young adults. Furowanin A (Fur A), one of the active ingredients of Millettia pachycarpa Benth, has been found to exert pro-apoptotic activity in human leukemia cells. This study is designed to evaluate the efficacy of Fur A against OS. The effect of Fur A on cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Western blotting and quantitative real-time PCR (qRT-PCR) were performed to determine the protein and mRNA level of sphingosine kinase 1 (SphK1), respectively. To validate the role of SphK1 in the pro-apoptotic activity of Fur A, overexpressing SphK1 vector and siRNA targeting SphK1 were utilized to transfect OS cells. Moreover, an OS xenograft murine model was used to analyze the therapeutic efficacy of Fur A in vivo. Fur A treatment led to a dose-dependent decrease in the number of viable cells. It also exhibited antiproliferative activity and significantly promoted apoptotic cell death in OS cell lines. Our results showed that the anticancer activity of Fur A was associated with downregulation of SphK1 and inactivation of its downstream signaling. The mediatory role of SphK1 was validated when the pro-apoptotic activity of Fur A was significantly blocked by SphK1 overexpression, while SphK1 knockdown sensitized the OS cells to Fur A. We concluded that Fur A can exhibit anti-growth and pro-apoptotic activities in vitro and in vivo in OS by downregulating SphK1. Our study highlights the possibility of utilizing Fur A as a chemotherapeutic agent in the treatment of OS. Anat Rec, 302:1941-1949, 2019. © 2019 American Association for Anatomy.

Significance of sphingosine kinase 1 expression in feline mammary tumors

BackgroundSphingosine kinase 1 (SPHK1) is an enzyme that converts pro-apoptotic ceramide and sphingosine into anti-apoptotic sphingosine-1-phosphate. There is growing evidence that SPHK1 activation promotes oncogenic transformation, tumor growth, chemotherapy resistance, and metastatic spread. High SPHK1 expression has been associated with a poor prognosis in several human cancers.ResultsIn the present study, the expression level of SPHK1 was examined in feline mammary tumor (FMT) specimens, and the IHC expression level of SPHK1 was associated with the histological grade of FMTs. IHC analysis of 88 FMT cases revealed that the expression level of SPHK1 was upregulated in 53 tumor tissues (60.2%) compared to adjacent mammary tissues. SPHK1 expression in FMTs was significantly associated with histological grade, presence of lymphovascular invasion, and estrogen receptor negativity. Treatment of primary FMT cells with SPHK1 inhibitors reduced cell viability, indicating that SPHK1 acts to promote FMT cell survival. These results indicate that SPHK1 may play an important role in FMTs and may be a therapeutic target in cats with FMT.ConclusionsSPHK1 over-expression in breast cancer tissues is associated with a poor prognosis in humans. SPHK1 over-expression in more aggressive FMTs provides support for a potential role of SPHK1 inhibitors for the treatment of FMTs. Targeting SPHK1 has potent cytotoxic effects in primary FMT cells. These findings suggest that further examination of the role SPHK1 plays in FMTs will pave the way for the investigation of SPHK1 inhibitors in future clinical applications.

The sphingosine kinase-1/sphingosine-1-phosphate axis in cancer: Potential target for anticancer therapy.

Sphingolipid metabolites, such as ceramide, sphingosine and sphingosine-1-phosphate (S1P), play many important roles in cellular activities. Ceramide and sphingosine inhibit cell proliferation and induce cell apoptosis while S1P has the opposite effect. Maintaining a metabolic balance of sphingolipids is essential for growth and development of cells. Sphingosine kinase (SPHK) is an important regulator for keeping this balance. It controls the level of S1P and plays important roles in proliferation, migration, and invasion of cancer cells and tumor angiogenesis. There are two isoenzymes of sphingosine kinase, SPHK1 and SPHK2. SPHK1 is ubiquitously expressed in most cancers where it promotes survival and proliferation, while SPHK2 is restricted to only certain tissues and its functions are not well characterized. SPHK1 is currently considered as a novel target for the treatment of cancers. Targeting SPHK1 would provide new strategies for cancer treatment and improve the prognosis of cancer patients. Here we review and summarize the current research findings on the SPHK1-S1P axis in cancer from many aspects including structure, expression, regulation, mechanism, and potential inhibitors.

Different Roles of Sphingosine Kinase 1 and 2 in Pancreatic Cancer Progression

BackgroundPancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. Materials and methodsS1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients. We generated PAN02 murine pancreatic cancer cell lines with a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system genes 9 (Cas9)-mediated deletion of SphK1 or SphK2 and assessed cell growth and migration. In an animal model, we assessed the survival of mice injected with PAN02 cells intraperitoneally. ResultsS1P levels in the pancreatic cancer tissue were significantly higher than those in noncancerous tissue. SphK1 knockout (KO) cells showed greater proliferation and migration than wild type (WT) cells, and SphK2 KO cells showed less proliferation and migration than WT cells. Animal experiments showed that the survival of mice injected with SphK1 KO cells was significantly shorter than those injected with WT cells, and the survival of mice injected with SphK2 KO cells was longer than those injected with WT cells. Surprisingly, cytotoxic assay using gemcitabine showed that SphK1 KO cells survived less than WT cells, and SphK2 KO cells survived more than WT cells. ConclusionsS1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cells. Targeting both SphK1 and SphK2 may be a potential strategy for pancreatic cancer treatment.

Physcion 8-O-β-glucopyranoside exhibits anti-leukemic activity through targeting sphingolipid rheostat.

Acute lymphoblastic leukemia (ALL) is the most common fatal cancer in people younger than 20 years of age. This study was designed to explore the anti-leukemia activity of physcion 8-O-β-glucopyranoside (PG) in B-cell ALL. NALM6 and SupB15 cells were used as model cell lines. Cell viability, cell apoptosis, cell cycle distribution were determined by CCK-8 assay, DNA fragmentation assay and flow cytometry, and flow cytometry, respectively. Expression of proteins involved in cell apoptosis and cell cycle regulation was determined by western blot and the levels of ceramide and sphingosine 1-phosphate (S1P) were determined by ELISA. Activity of sphingosine kinase 1 (SphK1) was also determined with a Sphingosine Kinase Assay Kit. In the present study, both model cell lines were transfected with siRNA targeting SphK1 or an overexpression plasmid to examine the role of SphK1 in the anti-leukemia activity of PG. Moreover, the efficacy of PG was examined in vivo in a mouse model by measuring survival and spleen weight. Our results provided experimental evidence that PG could significantly induce apoptosis and cell cycle arrest in vitro. Mechanistically, the anti-leukemia activity of PG was mediated by its ability to repress SphK1 and thus modulate ceramide-S1P rheostat. Moreover, the anti-leukemia activity of PG was also verified in a murine model. Collectively, our results indicate that PG may be a promising agent for the treatment of B-cell leukemia.

Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in an HER2 transgenic model.

Aberrant sphingolipid metabolism has been reported to promote breast cancer progression. Sphingosine kinase 1 (SphK1) is a key metabolic enzyme for the formation of pro-survival S1P from pro-apoptotic ceramide. The role of SphK1 in breast cancer has been well studied in estrogen receptor (ER)-positive breast cancer; however, its role in human epidermal growth factor 2 (HER2)-positive breast cancer remains unclear. Here, we show that genetic deletion of SphK1 significantly reduced mammary tumor development with reduced tumor incidence and multiplicity in the MMTV-neu transgenic mouse model. Gene expression analysis revealed significant reduction of claudin-2 (CLDN2) expression in tumors from SphK1 deficient mice, suggesting that CLDN2 may mediate SphK1's function. It is remarkable that SphK1 deficiency in HER2-positive breast cancer model inhibited tumor formation by the different mechanism from ER-positive breast cancer. In vitro experiments demonstrated that overexpression of SphK1 in ER-/PR-/HER2+ human breast cancer cells enhanced cell proliferation, colony formation, migration and invasion. Furthermore, immunostaining of SphK1 and CLDN2 in HER2-positive human breast tumors revealed a correlation in high-grade disease. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis. Targeting SphK1 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment.

Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings.

Abstract B17: Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model

Abstract Sphingosine kinase 1 (SphK1) phosphorylates pro-apoptotic sphingosine to form pro-survival sphingosine-1-phosphate (S1P), leading to cancer progression. In breast cancer, high levels of SphK1 mRNA are found in 80% of the patients, with average increase of 2-fold in breast tumors compared with normal tissue from the same patient. Recent studies suggest that SphK1 expression is associated with disease-free survival in ER-negative and HER2-positive breast tumors. In addition, inhibition of SphK1 in HER2-positive breast cancer cell line reduced S1P-induced HER2 and ERK1/2 activation. While these evidences suggest that SphK1 may play an important role in HER2-positive breast cancer, the exact role of SphK1 in breast tumorigenesis remains unclear. The aim of this project is to define the role of SphK1 in HER2-positive breast tumorigenesis and to investigate whether the pathway components are potential target for breast cancer prevention and therapies. Here, we report that SphK1 is required for HER2/neu-induced breast tumorigenesis and SphK1 regulates claudin-2 (CLDN2) to promote tumor development perhaps by mediating HER2. In MMTV-neu transgenic mouse model, genetic deletion of SphK1, which was accompanied by reduced S1P and increased C16-ceramide levels, significantly inhibited breast tumor development, with reduced incidence (SphK1-/-, 19%; SphK1+/-, 26%; and SphK1+/+, 57%; p < 0.05) and multiplicity (SphK1-/-, 0.27 ± 0.13; SphK1+/-, 0.32 ± 0.07; and SphK1+/+, 0.67 ± 0.14 tumors per animal; p < 0.05). Unexpectedly, SphK1 deletion did not affect tumor growth as results showed no differences in tumor volume. However, this is not surprising when considering the fact that the tumors were allowed to grow until tumor reached 20 mm in longer diameter, at which they were sacrificed regardless of age. Interestingly, whole genome expression analysis by Illumina MouseWG-6 v2.0 Expression BeadChip Array revealed significant reduction of CLDN2 expression in tumors from SphK1 deficient mice (p < 0.0001). In support to these findings, overexpression of SphK1 in MDA-MB-453 human breast cancer cells, which HER2 is amplified but lacks hormone receptors, lead to increase in CLDN2 expression (p < 0.05). Furthermore, overexpression of SphK1 in MDA-MB-453 cells also enhanced cell proliferation, colony formation, and migration/invasion, and drug resistance to cisplatin. The relevance of this finings in humans was confirmed by immunostaining of SphK1 and CLDN2 in HER2-positive breast cancer tumors and normal tissues (n = 93 and 35 subjects, respectively). The results showed that the expression of SphK1 was higher in tumors compared to adjacent normal tissue (p < 0.0001). Furthermore, higher SphK1 expression was observed in high grade tumors (vs low grade; p < 0.001) and ER-PR- tumors (vs ER+PR+; p < 0.0001). Interestingly, we observed similar staining pattern of CLDN2 proteins, where CLDN2 expression was high in tumors (p < 0.0001), particularly in high grade tumors (p < 0.01) and ER-PR- tumors (p < 0.0001). Moreover, we found a strong correlation between SphK1 and CLDN2 expressions in tumors but not in normal tissues, confirming the results obtained in animal study. Taken together, these findings suggest that SphK1 may play a pivotal role in HER2-positive breast carcinogenesis, and targeting SphK1 and/or CLDN2 may represent a novel approach for HER2-positive breast cancer chemoprevention and/or treatment. Citation Format: Yoshiko Shimizu, Hideki Furuya, Paulette M. Tamashiro, Steve Goodison, Owen Chan, Ian Pagano, Kayoko Iino, Rafael Peres, Kanani Hokutan, Lenora W.M. Loo, Brenda Hernandez, Aung Naing, Charles J. Rosser, Toshihiko Kawamori. Genetic deletion of sphingosine kinase 1 suppresses mouse breast tumor development in MMTV-neu transgenic model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B17.

The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines.

We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

Role of sphingosine kinase 1 and sphingosine-1-phosphate in CD40 signaling and IgE class switching.

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen-presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40-mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40-mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF-κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1(-/-) mice, isotype switching to antigen-specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40-mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen-specific IgE production.

Increased radiation sensitivity of head and neck squamous cell carcinoma with sphingosine kinase 1 inhibition

Sphingosine kinase 1 (SphK1) is an important regulator of apoptosis, survival, and proliferation in cancer cells. SphK1 expression in head and neck squamous cell cancer (HNSCC) cell lines and tumor tissue was assessed, and the efficacy of SphK1 knockdown in increasing tumor radiosensitivity was evaluated in vitro and in vivo. Expression of SphK1 was determined by immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) in 34 prospectively collected HNSCC tumor samples. HNSCC cell lines squamous cell carcinoma (SCC)-15 and SCC-25 were treated with SphK1 inhibitor SKI-II and siRNA targeting SphK1 with and without radiation, and the cell viability was assessed. SCC-15 cells with and without transfection of SphK1 siRNA were then injected into athymic nude mice to develop tumor xenografts, and these 2 groups were further divided into 1 group that received radiation and 1 group that did not. Tumor size was measured over 18 days, when the animals were killed and the tumors were evaluated by immunohistochemistry. SphK1 is found in both HNSCC cell lines and human tumor samples, with higher expression correlated with advanced tumor stage, nodal involvement, and recurrence. In vitro, both SCC-15 and SCC-25 were found to be radioresistant; however, they were sensitized by administration of SKI-II and transfection with siRNA targeting SphK1. In vivo, SphK1-siRNA transfected xenografts were decreased in size compared with both nonradiated control and radiated control mice, whereas mice with both SphK1-siRNA and radiation treatment showed a synergistic reduction in tumor volume. Histopathologic analysis demonstrated a decreased proliferative state in SphK1-siRNA transfected tumors. SphK1 is upregulated in HNSCC, and inhibition of SphK1 sensitizes HNSCC to radiation-induced cytotoxicity.

Targeting SphK1 as a new strategy against cancer.

Sphingolipid metabolites have emerged as critical players in a number of fundamental biological processes. Among them, sphingosine-1-phosphate (S1P) promotes cell survival and proliferation, in contrast to ceramide and sphingosine, which induce cell growth arrest and apoptosis. These sphingolipids with opposing functions are interconvertible inside cells, suggesting that a finely tuned balance between them can determine cell fate. Sphingosine kinases (SphKs), which catalyze the phosphorylation of sphingosine to S1P, are critical regulators of this balance. Of the two identified SphKs, sphingosine kinase type 1 (SphK1) has been shown to regulate various processes important for cancer progression and will be the focus of this review, since much less is known of biological functions of SphK2, especially in cancer. SphK1 is overexpressed in various types of cancers and upregulation of SphK1 has been associated with tumor angiogenesis and resistance to radiation and chemotherapy. Many growth factors, through their tyrosine kinase receptors (RTKs), stimulate SphK1 leading to a rapid increase in S1P. This S1P in turn can activate S1P receptors and their downstream signaling. Conversely, activation of S1P receptors can induce transactivation of various RTKs. Thus, SphK1 may play important roles in S1P receptor RTK amplification loops. Here we review the role of SphK1 in tumorigenesis, hormonal therapy, chemotherapy resistance, and as a prognostic marker. We will also review studies on the effects of SphK inhibitors in cells in vitro and in animals in vivo and in some clinical trials and highlight the potential of SphK1 as a new target for cancer therapeutics.

Cyclooxygenase-2 induction by adiponectin is regulated by a sphingosine kinase-1 dependent mechanism in cardiac myocytes

The adipose-derived plasma protein, adiponectin (APN), has various protective effects on cardiovascular diseases. In this study, we show that endogenous APN is required for full cyclooxygenase-2 (COX-2) induction by ischemia-reperfusion injury in the heart in vivo. In rat neonatal cardiac myocytes, APN-induced COX-2 expression was reduced by treatment with a sphingosine kinase-1 (SphK-1) inhibitor or siRNA targeting SphK-1. Treatment with a sphingosine-1-phosphate (S1P) receptor antagonist also diminished COX-2 expression in response to APN stimulation. These findings suggest that APN is a physiological regulator of COX-2 signaling in the heart and that this regulation occurs in part via a SphK-1–S1P receptor dependent mechanism in cardiac myocytes.