Targeting sphingosine kinase 1 in p53KO thymic lymphoma.

Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.

Upregulation of sphingosine kinase 1 in response to doxorubicin generates an angiogenic response via stabilization of Snail.

Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.

Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury.

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.

1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase.

Cancer cells may depend on exogenous serine, depletion of which results in slower growth and activation of adaptive metabolic changes. We previously demonstrated that serine and glycine (SG) deprivation causes loss of sphingosine kinase 1 (SK1) in cancer cells, thereby increasing the levels of its lipid substrate, sphingosine (Sph), which mediates several adaptive biological responses. However, the signaling molecules regulating SK1 and Sph levels in response to SG deprivation have yet to be defined. Here, we identify 1-deoxysphinganine (dSA), a noncanonical sphingoid base generated in the absence of serine from the alternative condensation of alanine and palmitoyl CoA by serine palmitoyl transferase, as a proximal mediator of SG deprivation in SK1 loss and Sph level elevation upon SG deprivation in cancer cells. SG starvation increased dSA levels in vitro and in vivo and in turn induced SK1 degradation through a serine palmitoyl transferase-dependent mechanism, thereby increasing Sph levels. Addition of exogenous dSA caused a moderate increase in intracellular reactive oxygen species, which in turn decreased pyruvate kinase PKM2 activity while increasing phosphoglycerate dehydrogenase levels, and thereby promoted serine synthesis. We further showed that increased dSA induces the adaptive cellular and metabolic functions in the response of cells to decreased availability of serine likely by increasing Sph levels. Thus, we conclude that dSA functions as an initial sensor of serine loss, SK1 functions as its direct target, and Sph functions as a downstream effector of cellular and metabolic adaptations. These studies define a previously unrecognized "physiological" nontoxic function for dSA.

A Milk-Fat Based Diet Increases Metastasis in the MMTV-PyMT Mouse Model of Breast Cancer.

A high-fat diet (HFD) and obesity are risk factors for many diseases including breast cancer. This is particularly important with close to 40% of the current adult population being overweight or obese. Previous studies have implicated that Mediterranean diets (MDs) partially protect against breast cancer. However, to date, the links between diet and breast cancer progression are not well defined. Therefore, to begin to define and assess this, we used an isocaloric control diet (CD) and two HFDs enriched with either olive oil (OOBD, high in oleate, and unsaturated fatty acid in MDs) or a milk fat-based diet (MFBD, high in palmitate and myristate, saturated fatty acids in Western diets) in a mammary polyomavirus middle T antigen mouse model (MMTV-PyMT) of breast cancer. Our data demonstrate that neither MFBD or OOBD altered the growth of primary tumors in the MMTV-PyMT mice. The examination of lung metastases revealed that OOBD mice exhibited fewer surface nodules and smaller metastases when compared to MFBD and CD mice. These data suggest that different fatty acids found in different sources of HFDs may alter breast cancer metastasis.

Loss of sphingosine kinase 1 increases lung metastases in the MMTV-PyMT mouse model of breast cancer.

Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.

Ceramide kinase regulates TNF-α-induced immune responses in human monocytic cells.

Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1ß and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.

The doxorubicin-induced cell motility network is under the control of the ceramide-activated protein phosphatase 1 alpha.

We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models.

Sphingosine kinase 1 downregulation is required for adaptation to serine deprivation.

It has been well-established that cancer cells often display altered metabolic profiles, and recent work has concentrated on how cancer cells adapt to serine removal. Serine can be either taken exogenously or synthesized from glucose, and its regulation forms an important mechanism for nutrient integration. One of the several important metabolic roles for serine is in the generation of bioactive sphingolipids since it is the main substrate for serine palmitoyltransferase, the initial and rate-limiting enzyme in the synthesis of sphingolipids. Previously, serine deprivation has been connected to the action of the tumor suppressor p53, and we have previously published on a role for p53 regulating sphingosine kinase 1 (SK1), an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). SK1 is a key enzyme in sphingolipid synthesis that functions in pro-survival and tumor-promoting pathways and whose expression is also often elevated in cancers. Here we show that SK1 was degraded during serine starvation in a time and dose-dependent manner, which led to sphingosine accumulation. This was independent of effects on p53 but required the action of the proteasome. Furthermore, we show that overexpression of SK1, to compensate for SK1 loss, was detrimental to cell growth under conditions of serine starvation, demonstrating that the suppression of SK1 under these conditions is adaptive. Mitochondrial oxygen consumption decreased in response to SK1 degradation, and this was accompanied by an increase in intracellular reactive oxygen species (ROS). Suppression of ROS with N-acteylcysteine resulted in suppression of the metabolic adaptations and in decreased cell growth under serine deprivation. The effects of SK1 suppression on ROS were mimicked by D-erythro-sphingosine, whereas S1P was ineffective, suggesting that the effects of loss of SK1 were due to the accumulation of its substrate sphingosine. This study reveals a new mechanism for regulating SK1 levels and a link of SK1 to serine starvation as well as mitochondrial function.

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides.

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence-as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated ß-galactosidase activity (SA-ß-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-ß-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.

Transcriptional Regulation of Sphingosine Kinase 1.

Once thought to be primarily structural in nature, sphingolipids have become increasingly appreciated as second messengers in a wide array of signaling pathways. Sphingosine kinase 1, or SK1, is one of two sphingosine kinases that phosphorylate sphingosine into sphingosine-1-phosphate (S1P). S1P is generally pro-inflammatory, pro-angiogenic, immunomodulatory, and pro-survival; therefore, high SK1 expression and activity have been associated with certain inflammatory diseases and cancer. It is thus important to develop an understanding of the regulation of SK1 expression and activity. In this review, we explore the current literature on SK1 transcriptional regulation, illustrating a complex system of transcription factors, cytokines, and even micro-RNAs (miRNAs) on the post transcriptional level.

Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α.

Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase-2 (nSMase2: SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1ß and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1ß and MCP-1. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF-α gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.

Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy.

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these "side" effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.

Yeast Sphingolipid Phospholipase Gene ISC1 Regulates the Spindle Checkpoint by a CDC55-Dependent Mechanism.

Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, and GIM4) as synthetically lethal with ISC1 The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, and KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1 Reciprocally, ISC1 deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions.

Delivery of long chain C16 and C24 ceramide in HeLa cells using oxidized graphene nanoribbons.

The bioactive sphingolipid ceramide has many important roles in cell signaling processes, particularly in signaling programmed cell death in cancer. However, ceramide levels are often impaired in multi-drug resistant and radiation resistant cancers due to the dysregulation of ceramide metabolism. Restoration of ceramide levels through external delivery therefore represents a potential therapeutic target for the treatment of resistant cancers. However, as a lipid, ceramide is extremely hydrophobic and requires a delivery system to enter cells. Here we report the development of a method to load significant amounts of the long chain C16 and C24 ceramides onto oxidized graphene nanoribbons (O-GNRs) derived from carbon nanotubes. Using O-GNRs as a delivery system for these ceramides, we were able to induce significant biological effects in HeLa cells in conjunction with C6 ceramide and ultraviolet radiation treatment. However, we found that O-GNRs themselves exert significant biological effects and can interfere with the actions of these ceramides and ultraviolet treatment. Loading of ceramides onto O-GNRs did not have a significant effect on the entry of the nanoparticles into cells. Despite the need for further improvement, these data represent an important first step in the development of O-GNRs as a delivery system for long chain ceramides.

Correction: PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

Mutational activation of the epidermal growth factor receptor (EGFR) is a major player in the pathogenesis of non-small cell lung cancer (NSCLC). NSCLC patients with constitutively active EGFR mutations eventually develop drug resistance against EGFR tyrosine-kinase inhibitors; therefore, better understandings of key components of mutant EGFR (mtEGFR) signaling are required. Here, we initially observed aberrantly high expression of protein kinase Cα (PKCα) in lung adenocarcinomas, especially those with EGFR mutations, and proceeded to examine the role of PKCα in the regulation of the signaling pathways downstream of mtEGFR. The results showed that NSCLC cell lines with constitutively active EGFR mutations tend to have very or moderately high PKCα levels. Furthermore, PKCα was constitutively activated in HCC827 and H4006 cells which have an EGFR deletion mutation in exon 19. Interestingly, mtEGFR was not required for the induction of PKCα at protein and message levels suggesting that the increased levels of PKCα are due to independent selection. On the other hand, mtEGFR activity was required for robust activation of PKCα. Loss of functions studies revealed that the NSCLC cells rely heavily on PKCα for the activation of the mTORC1 signaling pathway. Unexpectedly, the results demonstrated that PKCα was required for activation of Akt upstream of mTOR but only in cells with the mtEGFR and with the increased expression of PKCα. Functionally, inhibition of PKCα in HCC827 led to caspase-3-dependent apoptosis and a significant decrease in cell survival in response to cellular stress induced by serum starvation. In summary, the results identified important roles of PKCα in regulating mTORC1 activity in lung cancer cells, whereby a primary switching occurs from PKCα-independent to PKCα-dependent signaling in the presence of EGFR mutations. The results present PKCα as a potential synergistic target of personalized treatment for NSCLC with constitutively active mutant forms of EGFR and constitutively active PKCα.

Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases.

Sphingolipids contribute to the regulation of cell and tissue homeostasis, and disorders of sphingolipid metabolism lead to diseases such as inflammation, stroke, diabetes, and cancer. Sphingolipid metabolic pathways involve an array of enzymes that reside in specific subcellular organelles, resulting in the formation of many diverse sphingolipids with distinct molecular species based on the diversity of the ceramide (Cer) structure. In order to probe compartment-specific metabolism of sphingolipids in this study, we analyzed the Cer and SM species preferentially produced in the inner plasma membrane (PM), Golgi apparatus, ER, mitochondria, nucleus, and cytoplasm by using compartmentally targeted bacterial SMases and ceramidases. The results showed that the length of the acyl chain of Cer becomes longer according to the progress of Cer from synthesis in the ER to the Golgi apparatus, then to the PM. These findings suggest that each organelle shows different properties of SM-derived Cers consistent with its emerging distinct functions in vitro and in vivo.

Advances in determining signaling mechanisms of ceramide and role in disease.

Ceramide is a critical bioactive lipid involved in diverse cellular processes. It has been proposed to regulate cellular processes by influencing membrane properties and by directly interacting with effector proteins. Advances over the past decade have improved our understanding of ceramide as a bioactive lipid. Generation and characterization of ceramide-metabolizing enzyme KO mice, development of specific inhibitors and ceramide-specific antibodies, use of advanced microscopy and mass spectrometry, and design of synthetic ceramide derivatives have all provided insight into the signaling mechanisms of ceramide and its implications in disease. As a result, the role of ceramide in biological functions and disease are now better understood, with promise for development of therapeutic strategies to treat ceramide-regulated diseases.

Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis.

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.