High-fat diet (HFD) intake has been linked to an increased risk of pancreatic ductal adenocarcinoma (PDAC), a lethal and therapy-resistant cancer. However, whether and how specific dietary fats drive cancer development remains unresolved. Leveraging an oncogenic Kras -driven mouse model that closely mimics human PDAC progression, we screened a dozen isocaloric HFDs differing solely in fat source and representing the diversity of human fat consumption. Unexpectedly, diets rich in oleic acid - a monounsaturated fatty acid (MUFA) typically associated with good health - markedly enhanced tumorigenesis. Conversely, diets high in polyunsaturated fatty acids (PUFAs) suppressed tumor progression. Relative dietary fatty acid saturation levels (PUFA/MUFA) governed pancreatic membrane phospholipid composition, lipid peroxidation, and ferroptosis sensitivity in mice, concordant with circulating PUFA/MUFA levels being linked to altered PDAC risk in humans. These findings directly implicate dietary unsaturated fatty acids in controlling ferroptosis susceptibility and tumorigenesis, supporting potential "precision nutrition" strategies for PDAC prevention.

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite's ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin–streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied dithionite quenching to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

Deoxysphingolipids (dSLs) are atypical sphingolipids that accumulate in several pathological settings, yet their impact on hematologic malignancies is poorly understood. Here, we investigate the pathways and mechanisms of deoxysphinganine (dSA) cytotoxicity in lymphoma cells and its potential as a therapeutic agent. dSA exhibited markedly greater cytotoxicity than canonical sphingoid bases in lymphoma cell lines, yet induced only cytostatic effects in normal human T cells, indicating a therapeutically exploitable window. Inhibition of ceramide synthase blocked the generation of deoxy(dihydro)ceramides, prevented mitochondrial depolarization, caspase activation, ER stress, and DNA damage, establishing CerS-dependent deoxysphingolipids as essential mediators of dSA-induced death. Mechanistically, dSA engaged a mitochondrial apoptotic pathway, with DNA damage occurring downstream of mitochondrial permeabilization and caspase activation, while PERK-driven ER stress occurred in parallel and was dispensable for cytotoxicity. Subtype-specific engagement of ER stress and DNA damage further suggests that dSL signaling is shaped by lineage context. The differential sensitivity between malignant lymphoid cells and normal T cells, together with the central role of CerS-derived deoxy(dihydro)ceramides, highlights deoxysphingolipid metabolism as a druggable vulnerability in lymphoma. These findings support further exploration of dSA-based strategies and targeted modulation of dSL synthesis as a novel therapeutic avenue for non-solid hematologic malignancies.

Cancer cells must adapt to harsh tumor microenvironments, including acidic stress, to survive and thrive. Understanding how cancer cells achieve this adaptation can uncover new biomarkers and therapeutic strategies. In this study, we investigated the spatial metabolic phenotypic heterogeneity of breast cancer cells in acidic habitats using spatial multi-omics approaches on 3D spheroids. We found that cancer cells dynamically regulate sphingolipid metabolism to fine-tune their cell state to cope with acidic selection pressures. Cancer cells evolve mechanisms to deal with initially accumulating toxic ceramides but later adapt to it by rerouting SL metabolic pathways to eliminate them. Using advanced MALDI image analysis, and SL inhibitors on patient derived organoids, we demonstrated that cancer cells can switch between metabolic routes when key pathways are blocked, showcasing remarkable cell state plasticity. These insights highlight the potential to target metabolic plasticity as a novel therapeutic strategy to disrupt cancer adaptation and evolution, offering new avenues for cancer treatment.

Sphingolipids (SPLs) are bioactive lipids playing vital functions in cellular stress responses. The tumor suppressor p53 has been implicated in regulating a few specific SPL enzymes; however, a comprehensive understanding of p53’s overall impact on SPL metabolism is lacking. Here, we employed an integrative biochemical approach combining a novel flux tracing method (using d17dihydrosphingosine) with in situ enzymatic activity assays in the context of treatment with doxorubicin (Dox), a DNA-damaging agent causing well-established dose-dependent activation of p53. Furthermore, our previous studies established dose-specific modulation of SPLs by sublethal (low dose) versus lethal (high dose) Dox. Here, we exploited this model to focus on the role of p53, and found i) both low and high Dox enhanced the rate of synthesis of select dihydroceramide species, d17:0/16:0, d17:0/18:0, and d17:0/20:0, implicating activation of specific ceramide (Cer) synthases (1/4 and 5/6), with p53 dependence only at low Dox (LD); ii) novel p53-dependent suppression of dihydroceramide desaturase activity at both Dox doses; iii) both doses of Dox impaired the synthesis of d17hexosylceramide and d17sphingomyelin, with an unanticipated role for p53 only at LD; and iv) with respect to inhibition of d17sphingomyelin synthesis, an investigation into Cer transport to the Golgi identified the Cer transport protein (CERT1) as a novel target of Dox (reduction of protein and activity) and p53 (reduction of activity, particularly at LD). These observations underscore p53's prominent role as a master regulator of SPL metabolism, inducing major remodeling of cellular SPL metabolism with extensive and integrated effects on SPL synthesis.

The increasing interest in the role of sphingolipids in (patho)physiology has led to the demand for visualization of these lipids within tissue samples (both from animal models and patient specimens) using techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While increasingly adopted, detection of sphingolipids with MALDI-MSI is challenging due to: i) the significant structural variations of sphingolipid molecules, ii) the potential breakdown of the more complex molecules into structurally simpler species which may confound the analysis, and iii) the great difference in levels among sphingolipid classes and subspecies, with the low-abundant ones often being close to the detection limit. In this study, we adopted a multi-pronged approach to establish a robust pipeline for the detection of sphingolipids by MALDI-MSI and to establish best practices and limitations of this technology. First, we evaluated the more commonly adopted methods [2,5-Dihydroxyacetophenon (DHA) or 2,5-Dihydroxybenzoic acid (DHB) matrix in positive ion mode and 1,5-Diaminonaphthalene (DAN) matrix in negative ion mode] using MALDI-MS on reference standards. These standards were used at ratios similar to their relative levels in biological samples to evaluate signal artifacts originating from fragmentation of more complex sphingolipids and impacting low level species. Next, by applying the most appropriate protocol for each sphingolipid class, MALDI-MSI signals were validated in cell culture by modulating specific sphingolipid species using sphingolipid enzymes and inhibitors. Finally, the optimized parameters were utilized on breast cancer tissue from the PyMT mouse model. We report the optimal signal for sphingomyelin (SM) and, for the first time, Sph in DHB positive ion mode (in cells and PyMT tissue), and the validated detection of ceramides and glycosphingolipids in DAN negative ion mode. We document the extensive fragmentation of SM into sphingosine-1-phosphate (S1P) and even more so into ceramide-1-phosphate (C1P) using DAN in negative ion mode and its effect in generating an artifactual C1P tissue signal; we also report the lack of detectable signal for S1P and C1P in biological samples (cells and tissue) using the more suitable DHB positive ion mode protocol.

Hereditary spastic paraplegia subtype SPG54 is a genetic neurological disorder caused by mutations in the DDHD2 gene. Excessive lipid droplet accumulation is observed in the brains of SPG54 patients and DDHD2 knockout mice, consistent with DDHD2’s reported neutral lipase activity. Here, we find recombinant human DDHD2 preferentially hydrolyzes diacylglycerol (DAG) over phospholipids, with a slight preference for DAG over triacylglycerol (TAG). DDHD2 also exhibits transacylase activity, which enables transfer of acyl chains from TAGs to DAGs and monoacylglycerols to remodel the acyl chains of TAGs. A predicted hydrophobic amphipathic helix on DDHD2 is essential for lipid droplet binding in vitro and in cells, and its lack reduces the enzymatic activity and TAG acyl chain remodeling. Adipose triglyceride lipase, but not hormone sensitive lipase, also has transacylation activity and can remodel TAG acyl chains, but to a lesser extent than DDHD2. Taken together, this provides evidence that DDHD2 is a neutral lipid lipase and transacylase whose broad specificity enables TAG acyl-chain remodeling.

Although Doxorubicin (Dox) is an effective chemotherapeutic, its clinical utility is limited by a cumulative dose-dependent cardiotoxicity. While mechanisms underlying this cardiotoxicity have been investigated, strategies targeting these pathways have had marginal effects or had potential to interfere with Dox's anti-cancer activity. Sphingolipids (SL) are central to the chemotherapy response in multiple cancers, yet comparatively little is known about their role in non-transformed tissue, and actionable SL targets have not been identified. Here, we identified the SL enzyme neutral sphingomyelinase-2 (nSMase2) as a crucial downstream effector of Dox that is critical for chronic Dox-induced cardiotoxicity. In vitro studies showed that Dox treatment induces nSMase2 mRNA, protein, activity, and Cer accumulation in cardiomyocytes (CM) but not in cardiac fibroblasts. Mechanistically, nSMase2 induction was downstream of Top2B and p53, two previously identified molecular regulators of Dox-induced cardiotoxicity. In vivo studies in a chronic Dox model of cardiotoxicity found that loss of nSMase2 activity-null fro/fro mice were significantly protected from Dox-induced cardiac damage, exhibiting maintained ejection fraction, fractional shortening, and reduced left ventricle mass compared to wild-type littermates. Biologically, nSMase2 was dispensable for Dox-induced cell death but was important for Dox-induced CM senescence both in vitro and in vivo . Microarray analysis identified the dual specificity phosphatase DUSP4 as a downstream target of nSMase2 in vitro in Dox-treated CMs and in vivo in the chronic Dox-treated heart. Taken together, these results establish nSMase2 as a key component of the DNA damage response pathway in CMs and define a critical role for nSMase2 as a SL mediator of Dox-induced cardiotoxicity through effects on CM senescence. In addition to cementing a role for SLs in Dox effects in normal tissue, this study further advances nSMase2 as a target of interest for cardioprotection.

Acyltransferase enzymes (EC 2.3.) are a large group of enzymes that transfer acyl groups to a variety of substrates. This review focuses on fatty acyltransferases involved in the biosynthetic pathways of glycerolipids and sphingolipids and how these enzymes have been pharmacologically targeted in their biologic context. Glycerolipids and sphingolipids, commonly treated independently in their regulation and biologic functions, are put together to emphasize the parallelism in their metabolism and bioactive roles. Furthermore, a newly considered signaling molecule, 1-O-acylceramide, resulting from the acylation of ceramide by DGAT2 enzyme, is discussed. Finally, the implications of DGAT2 as a putative ceramide acyltransferase (CAT) enzyme, with a putative dual role in TAG and 1-O-acylceramide generation, are explored. SIGNIFICANCE STATEMENT This manuscript reviews the current status of drug development in lipid acyltransferases. These are current targets in metabolic syndrome and other diseases, including cancer. A novel function for a member in this group of lipids has been recently reported in cancer cells. The responsible enzyme and biological implications of this added member are discussed.

SPTLC3, an inducible subunit of the serine palmitoyltransferase (SPT) complex, causes production of alternative sphingoid bases, including a 16-carbon dihydrosphingosine, whose biological function is only beginning to emerge. High-fat feeding induced SPTLC3 in the liver, prompting us to produce a liver-specific knockout mouse line. Following high-fat feeding, knockout mice showed decreased fasting blood glucose, and knockout primary hepatocytes showed suppressed glucose production, a core function of hepatocytes. Stable isotope tracing revealed suppression of the gluconeogenic pathway, finding that SPTLC3 was required to maintain expression of key gluconeogenic genes via adenylate cyclase/cyclic AMP (cAMP)/cAMP response element binding protein (CREB) signaling. Additionally, by employing a combination of a recently developed lipidomics methodology, exogenous C14/C16 fatty acid treatment, and in situ adenylate cyclase activity, we implicated a functional interaction between sphingomyelin with a d16 backbone and adenylate cyclase at the plasma membrane. This work pinpoints a specific sphingolipid-protein functional interaction with broad implications for understanding sphingolipid signaling and metabolic disease.

During the last 30 years, an increasing number of cellular functions have been reported to be regulated by the lipid ceramide. The diversity in the ceramide structure, leading to tens of ceramide species and the discrete distribution based on subcellular topology, could explain the wide variety of functions attributed to this bioactive lipid. One of these pools of ceramide resides in the plasma membrane, and several works have suggested that an increase in plasma membrane ceramide (PMCer) in response to stimulation leads to cell death and modulates cell adhesion and migration. However, there is a limitation in studying PMCer content in this location primarily due to the inability to quantify its mass. Our group recently developed a method to specifically quantitate PMCer. In this work, we interrogate what sphingolipid metabolizing enzymes are responsible for modulating the basal levels of plasma membrane ceramide. An in-silico prediction and experimental confirmation found an almost perfect correlation between the endogenous expression levels of neutral sphingomyelinase (nSMase2) and the amount of plasma membrane ceramide in unstimulated cells. Manipulating the expression levels of nSMase2, but not other candidate enzymes of ceramide metabolism, profoundly affected PMCer. Moreover, a physiologic induction of nSMase2 during cell confluence resulted in a nSMase2-dependent dramatic increase in PMCer. Together, these results identify nSMase2 as the primary enzyme to regulate plasma membrane ceramide.

Sphingolipids (SLs) constitute a discrete subdomain of metabolism, and they display both structural and signaling functions. Accumulating evidence also points to intimate connections between intermediary metabolism and SL metabolism. Given that many SLs exhibit bioactive properties (i.e. transduce signals), these raise the possibility that an important function of SLs is to relay information on metabolic changes into specific cell responses. This could occur at various levels. Some metabolites are incorporated into SLs, whereas others may initiate regulatory or signaling events that, in turn, modulate SL metabolism. In this review, we elaborate on the former as it represents a poorly appreciated aspect of SL metabolism, and we develop the hypothesis that the SL network is highly sensitive to several specific metabolic changes, focusing on amino acids (serine and alanine), various fatty acids, choline (and ethanolamine), and glucose.

Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the US and is anticipated to become the second within the next decade. Epidemiologic studies support a strong link between high fat diet (HFD) consumption and increased PDAC risk, however, the translational relevance of diet research has been limited by inconsistencies in fat source and consumption across human populations and mouse studies. Therefore, whether and how specific dietary fatty acids drive cancer development is poorly understood. To address this gap in knowledge, we performed a diet screen, comprised of 12 isocaloric HFDs differing solely in fat source and representing the diversity of modern human fat consumption, in an oncogenic Kras-driven mouse model (KC: Kras LSL-G12D /+;Pdx1-Cre) that closely mimics the genetic and histologic features of human PDAC progression. Unexpectedly, we discovered that diets rich in oleic acid – a monounsaturated fatty acid (MUFA) typically associated with good health – enhanced pancreatic tumorigenesis. Furthermore, we observed a strong positive correlation between increased tumor burden and diets exhibiting a high ratio of MUFAs to polyunsaturated fatty acids (PUFAs), concordant with high MUFA to PUFA levels in the plasma and in pancreatic phospholipids. Membrane phospholipid incorporation of MUFAs (relative to PUFAs) suppresses lipid peroxidation and cell death via ferroptosis. Consistent with this, pancreata from mice fed a MUFA-rich diet showed decreased expression of genes involved in lipid peroxidation and reduced histologic evidence of lipid peroxidation end products compared to a PUFA-rich diets. Furthermore, primary acinar cells from mice fed high MUFA diets were resistant to cell death upon treatment with ferroptosis inhibitors. Conversely, diets rich in PUFAs increased PUFA incorporation into pancreatic phospholipids, activated transcriptional programs of lipid peroxidation, and intercepted Kras-driven tumor progression in vivo. Collectively, these data support a role for dietary fatty acids in molding the pancreatic phospholipid landscape, altering lipid peroxidation and ferroptosis sensitivity to dictate tumor fate. Citation Format: Christian F Ruiz, Xiangyu Ge, Rylee McDonnell, Daniel McQuaid, Jennifer Kaplan, Guangtao Li, Michael C Rudolph, Fred F Gorelick, John Wysolmerski, Daniel Canals, John D Haley, Matthew Rodeheffer, Mandar D Muzumdar. Dietary fats dictate pancreatic cancer fate via phospholipid saturation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-06.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

Over the past 30 years, a growing body of evidence has revealed the regulatory role of the lipid ceramide in various cellular functions. The structural diversity of ceramide, resulting in numerous species, and its distinct distribution within subcellular compartments may account for its wide range of functions. However, our ability to study the potential role of ceramide in specific subcellular membranes has been limited. Several works have shown mitochondrial, Golgi, and plasma membrane ceramide to mediate signaling pathways independently. These results have started to shift the focus on ceramide signaling research toward specific membrane pools. Nonetheless, the challenge arises from the substantial intracellular ceramide content, hindering efforts to quantify its presence in particular membranes. Recently, we have developed the first method capable of detecting and quantifying ceramide in the plasma membrane, leading to unexpected results such as detecting different pools of ceramide responding to drug concentration or time. This review summarizes the historical context that defined the idea of pools of ceramide, the studies on plasma membrane ceramide as a bioactive entity, and the tools available for its study, especially the new method to detect and, for the first time, quantify plasma membrane ceramide. We believe this method will open new avenues for researching sphingolipid signaling and metabolism.