TGIF transcription factors repress acetyl CoA metabolic gene expression and promote intestinal tumor growth.

Tgif1 (thymine-guanine-interacting factor 1) and Tgif2 repress gene expression by binding directly to DNA or interacting with transforming growth factor (TGF) ß-responsive SMADs. Tgifs are essential for embryogenesis and may function in tumor progression. By analyzing both gain and loss of Tgif function in a well-established mouse model of intestinal cancer, we show that Tgifs promote adenoma growth in the context of mutant Apc (adenomatous polyposis coli). Despite the tumor-suppressive role of TGFß signaling, transcriptome profiling of colon tumors suggests minimal effect of Tgifs on the TGFß pathway. Instead, it appears that Tgifs, which are up-regulated in Apc mutant colon tumors, contribute to reprogramming metabolic gene expression. Integrating gene expression data from colon tumors with other gene expression and chromatin-binding data identifies a set of direct Tgif target genes encoding proteins involved in acetyl CoA and pyruvate metabolism. Analysis of both tumor and nontumor tissues indicates that these genes are targets of Tgif repression in multiple settings, suggesting that this is a core Tgif function. We propose that Tgifs play an important role in regulating basic energy metabolism in normal cells, and that this function of Tgifs is amplified in some cancers.

Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Macrophage metabolic adaptation to heme detoxification involves CO-dependent activation of the pentose phosphate pathway.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase-dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.

Nervonic acid limits weight gain in a mouse model of diet-induced obesity.

Lipid perturbations contribute to detrimental outcomes in obesity. We previously demonstrated that nervonic acid, a C24:1 ω-9 fatty acid, predominantly acylated to sphingolipids, including ceramides, are selectively reduced in a mouse model of obesity. It is currently unknown if deficiency of nervonic acid-sphingolipid metabolites contribute to complications of obesity. Mice were fed a standard diet, a high fat diet, or these diets supplemented isocalorically with nervonic acid. The primary objective was to determine if dietary nervonic acid content alters the metabolic phenotype in mice fed a high fat diet. Furthermore, we investigated if nervonic acid alters markers of impaired fatty acid oxidation in the liver. We observed that a nervonic acid-enriched isocaloric diet reduced weight gain and adiposity in mice fed a high fat diet. The nervonic acid enrichment led to increased C24:1-ceramides and improved several metabolic parameters including blood glucose levels, and insulin and glucose tolerance. Mechanistically, nervonic acid supplementation increased PPARα and PGC1α expression and improved the acylcarnitine profile in liver. These alterations indicate improved energy metabolism through increased ß-oxidation of fatty acids. Taken together, increasing dietary nervonic acid improves metabolic parameters in mice fed a high fat diet. Strategies that prevent deficiency of, or restore, nervonic acid may represent an effective strategy to treat obesity and obesity-related complications.

Glucosylceramide production maintains colon integrity in response to Bacteroides fragilis toxin-induced colon epithelial cell signaling.

Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro-inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E-cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT-induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide-dependent mechanism by which the colon epithelium responds to BFT.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Acid ceramidase promotes drug resistance in acute myeloid leukemia through NF-κB-dependent P-glycoprotein upregulation.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

Conductance-Based Biophysical Distinction and Microfluidic Enrichment of Nanovesicles Derived from Pancreatic Tumor Cells of Varying Invasiveness.

Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.

Sphingosine 1-Phosphate Lyase Enhances the Activation of IKKε To Promote Type I IFN-Mediated Innate Immune Responses to Influenza A Virus Infection.

Sphingosine 1-phosphate (S1P) lyase (SPL) is an intracellular enzyme that mediates the irreversible degradation of the bioactive lipid S1P. We have previously reported that overexpressed SPL displays anti-influenza viral activity; however, the underlying mechanism is incompletely understood. In this study, we demonstrate that SPL functions as a positive regulator of IKKε to propel type I IFN-mediated innate immune responses against viral infection. Exogenous SPL expression inhibited influenza A virus replication, which correlated with an increase in type I IFN production and IFN-stimulated gene accumulation upon infection. In contrast, the lack of SPL expression led to an elevated cellular susceptibility to influenza A virus infection. In support of this, SPL-deficient cells were defective in mounting an effective IFN response when stimulated by influenza viral RNAs. SPL augmented the activation status of IKKε and enhanced the kinase-induced phosphorylation of IRF3 and the synthesis of type I IFNs. However, the S1P degradation-incompetent form of SPL also enhanced IFN responses, suggesting that SPL's pro-IFN function is independent of S1P. Biochemical analyses revealed that SPL, as well as the mutant form of SPL, interacts with IKKε. Importantly, when endogenous IKKε was downregulated using a small interfering RNA approach, SPL's anti-influenza viral activity was markedly suppressed. This indicates that IKKε is crucial for SPL-mediated inhibition of influenza virus replication. Thus, the results illustrate the functional significance of the SPL-IKKε-IFN axis during host innate immunity against viral infection.

A MicroRNA93-Interferon Regulatory Factor-9-Immunoresponsive Gene-1-Itaconic Acid Pathway Modulates M2-Like Macrophage Polarization to Revascularize Ischemic Muscle.

BACKGROUND: Currently, no therapies exist for treating and improving outcomes in patients with severe peripheral artery disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and to reduce tissue loss in genetic PAD models. However, the cell-specific function, downstream mechanisms, or signaling involved in miR93-mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD, and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1 activation/polarization state. In the present study, we identified a novel mechanism by which miR93 regulates macrophage polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental PAD. METHODS: In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation conditions) and in vivo experiments in preclinical PAD models (unilateral femoral artery ligation and resection) were conducted to examine the role of miR93-interferon regulatory factor-9-immunoresponsive gene-1 (IRG1)-itaconic acid pathway in macrophage polarization, angiogenesis, arteriogenesis, and perfusion recovery. RESULTS: In vivo, compared with wild-type controls, miR106b-93-25 cluster-deficient mice (miR106b-93-25-/-) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like macrophages after experimental PAD. Intramuscular delivery of miR93 in miR106b-93-25-/- PAD mice increased angiogenesis, arteriogenesis, and the extent of perfusion, which correlated with more M2-like macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like polarization even under M1-like polarizing conditions (hypoxia serum starvation). Delivery of bone marrow-derived macrophages from miR106b-93-25-/- to wild-type ischemic muscle decreased angiogenesis, arteriogenesis, and perfusion, whereas transfer of wild-type macrophages to miR106b-93-25-/- had the opposite effect. Systematic analysis of top differentially upregulated genes from RNA sequencing between miR106b-93-25-/- and wild-type ischemic muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage polarization. The 3' untranslated region luciferase assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not an miR93 target but that interferon regulatory factor-9, which can regulate IRG1 expression, is an miR93 target. In vitro, increased expression of interferon regulatory factor-9 and IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential. CONCLUSIONS: miR93 inhibits interferon regulatory factor-9 to decrease IRG1-itaconic acid production to induce M2-like polarization in ischemic muscle to enhance angiogenesis, arteriogenesis, and perfusion recovery in experimental PAD.

Gut microbiota dysbiosis is associated with malnutrition and reduced plasma amino acid levels: Lessons from genome-scale metabolic modeling.

Malnutrition is a severe non-communicable disease, which is prevalent in children from low-income countries. Recently, a number of metagenomics studies have illustrated associations between the altered gut microbiota and child malnutrition. However, these studies did not examine metabolic functions and interactions between individual species in the gut microbiota during health and malnutrition. Here, we applied genome-scale metabolic modeling to model the gut microbial species, which were selected from healthy and malnourished children from three countries. Our analysis showed reduced metabolite production capabilities in children from two low-income countries compared with a high-income country. Additionally, the models were also used to predict the community-level metabolic potentials of gut microbes and the patterns of pairwise interactions among species. Hereby we found that due to bacterial interactions there may be reduced production of certain amino acids in malnourished children compared with healthy children from the same communities. To gain insight into alterations in the metabolism of malnourished (stunted) children, we also performed targeted plasma metabolic profiling in the first 2 years of life of 25 healthy and 25 stunted children. Plasma metabolic profiling further revealed that stunted children had reduced plasma levels of essential amino acids compared to healthy controls. Our analyses provide a framework for future efforts towards further characterization of gut microbial metabolic capabilities and their contribution to malnutrition.

Liposomal Delivery of Diacylglycerol Lipase-Beta Inhibitors to Macrophages Dramatically Enhances Selectivity and Efficacy in Vivo.

Diacylglycerol lipase-beta (DAGLß) hydrolyzes arachidonic acid (AA)-containing diacylglycerols to produce bioactive lipids including endocannabinoids and AA-derived eicosanoids involved in regulation of inflammatory signaling. Previously, we demonstrated that DAGLß inactivation using the triazole urea inhibitor KT109 blocked macrophage inflammatory signaling and reversed allodynic responses of mice in inflammatory and neuropathic pain models. Here, we tested whether we could exploit the phagocytic capacity of macrophages to localize delivery of DAGLß inhibitors to these cells in vivo using liposome encapsulated KT109. We used DAGLß-tailored activity-based probes and chemical proteomic methods to measure potency and selectivity of liposomal KT109 in macrophages and tissues from treated mice. Surprisingly, delivery of ∼5 µg of liposomal KT109 was sufficient to achieve ∼80% inactivation of DAGLß in macrophages with no apparent activity in other tissues in vivo. Our macrophage-targeted delivery resulted in a >100-fold enhancement in antinociceptive potency compared with free compound in a mouse inflammatory pain model. Our studies describe a novel anti-inflammatory strategy that is achieved by targeted in vivo delivery of DAGLß inhibitors to macrophages.

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia.

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers.

Lysosomal degradation of CD44 mediates ceramide nanoliposome-induced anoikis and diminished extravasation in metastatic carcinoma cells.

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.

Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia--Impact on enzyme activity and response to cytotoxics.

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N-desmethyltamoxifen (DMT), block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 µM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme-catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT (i) increased the levels of endogenous C16:0 and C24:1 ceramide molecular species, (ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, (iii) drastically reduced levels of sphingosine (to 9% of control), and (iv) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. The co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug-resistant setting.

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.

Preclinical development of a C6-ceramide NanoLiposome, a novel sphingolipid therapeutic.

Despite the therapeutic potential of sphingolipids, the ability to develop this class of compounds as active pharmaceutical ingredients has been hampered by issues of solubility and delivery. Beyond these technical hurdles, significant challenges in completing the necessary preclinical studies to support regulatory review are necessary for commercialization. This review seeks to identify the obstacles and potential solutions in the translation of a novel liposomal technology from the academic bench to investigational new drug (IND) stage by discussing the preclinical development of the Ceramide NanoLiposome (CNL), which is currently being developed as an anticancer drug for the initial indication of hepatocellular carcinoma (HCC).

Ceramide 1-phosphate mediates endothelial cell invasion via the annexin a2-p11 heterotetrameric protein complex.

The bioactive sphingolipid, ceramide 1-phosphate (C-1-P), has been implicated as an extracellular chemotactic agent directing cellular migration in hematopoietic stem/progenitor cells and macrophages. However, interacting proteins that could mediate these actions of C-1-P have, thus far, eluded identification. We have now identified and characterized interactions between ceramide 1-phosphate and the annexin a2-p11 heterotetramer constituents. This C-1-P-receptor complex is capable of facilitating cellular invasion. Herein, we demonstrate in both coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells that C-1-P induces invasion through an extracellular matrix barrier. By employing surface plasmon resonance, lipid-binding ELISA, and mass spectrometry technologies, we have demonstrated that the heterotetramer constituents bind to C-1-P. Although the annexin a2-p11 heterotetramer constituents do not bind the lipid C-1-P exclusively, other structurally similar lipids, such as phosphatidylserine, sphingosine 1-phosphate, and phosphatidic acid, could not elicit the potent chemotactic stimulation observed with C-1-P. Further, we show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular annexin a2-p11 heterotetrameric protein, can mediate vascular endothelial cell invasion.

Novel off-target effect of tamoxifen--inhibition of acid ceramidase activity in cancer cells.

Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mitogenic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit micromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with tamoxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5min, and dose-dependent, as low as 5µM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH4.5. In addition, pretreatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism.