A fluorogenic substrate for the detection of lipid amidases in intact cells.

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 µM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 µM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 µM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.

Monogenic neurological disorders of sphingolipid metabolism.

Sphingolipids comprise a wide variety of molecules containing a sphingoid long-chain base that can be N-acylated. These lipids are particularly abundant in the central nervous system, being membrane components of neurons as well as non-neuronal cells. Direct evidence that these brain lipids play critical functions in brain physiology is illustrated by the dramatic consequences of genetic disturbances of their metabolism. Inherited defects of both synthesis and catabolism of sphingolipids are now identified in humans. These monogenic disorders are due to mutations in the genes encoding for the enzymes that catalyze either the formation or degradation of simple sphingolipids such as ceramides, or complex sphingolipids like glycolipids. They cause varying degrees of central nervous system dysfunction, quite similarly to the neurological disorders induced in mice by gene disruption of the corresponding enzymes. Herein, the enzyme deficiencies and metabolic alterations that underlie these diseases are reviewed. Their possible pathophysiological mechanisms and the functions played by sphingolipids one can deduce from these conditions are discussed. This article is part of a Special Issue entitled Brain Lipids.

Spinal muscular atrophy associated with progressive myoclonic epilepsy is caused by mutations in ASAH1.

Spinal muscular atrophy (SMA) is a clinically and genetically heterogeneous disease characterized by the degeneration of lower motor neurons. The most frequent form is linked to mutations in SMN1. Childhood SMA associated with progressive myoclonic epilepsy (SMA-PME) has been reported as a rare autosomal-recessive condition unlinked to mutations in SMN1. Through linkage analysis, homozygosity mapping, and exome sequencing in three unrelated SMA-PME-affected families, we identified a homozygous missense mutation (c.125C>T [p.Thr42Met]) in exon 2 of ASAH1 in the affected children of two families and the same mutation associated with a deletion of the whole gene in the third family. Expression studies of the c.125C>T mutant cDNA in Farber fibroblasts showed that acid-ceramidase activity was only 32% of that generated by normal cDNA. This reduced activity was able to normalize the ceramide level in Farber cells, raising the question of the pathogenic mechanism underlying the CNS involvement in deficient cells. Morpholino knockdown of the ASAH1 ortholog in zebrafish led to a marked loss of motor-neuron axonal branching, a loss that is associated with increased apoptosis in the spinal cord. Our results reveal a wide phenotypic spectrum associated with ASAH1 mutations. An acid-ceramidase activity below 10% results in Farber disease, an early-onset disease starting with subcutaneous lipogranulomata, joint pain, and hoarseness of the voice, whereas a higher residual activity might be responsible for SMA-PME, a later-onset phenotype restricted to the CNS and starting with lower-motor-neuron disease.

Human genetic disorders of sphingolipid biosynthesis.

Monogenic defects of sphingolipid biosynthesis have been recently identified in human patients. These enzyme deficiencies affect the synthesis of sphingolipid precursors, ceramides or complex glycosphingolipids. They are transmitted as autosomal recessive or dominant traits, and their resulting phenotypes often replicate the abnormalities seen in murine models deficient for the corresponding enzymes. In quite good agreement with the known critical roles of sphingolipids in cells from the nervous system and the epidermis, these genetic defects clinically manifest as neurological disorders, including paraplegia, epilepsy or peripheral neuropathies, or present with ichthyosis. The present review summarizes the genetic alterations, biochemical changes and clinical symptoms of this new group of inherited metabolic disorders. Hypotheses regarding the molecular pathophysiology and potential treatments of these diseases are also discussed.

The nonlysosomal ß-glucosidase GBA2 promotes endoplasmic reticulum stress and impairs tumorigenicity of human melanoma cells.

Glycosphingolipids, which are abundant at the surface of melanoma cells, play crucial roles in tumor progression. We investigated whether a newly described glycosphingolipid hydrolase, encoded by the GBA2 gene, can modulate human melanoma cell growth and death. GBA2 expression was quantified on melanoma cells by RT-qPCR. The antiproliferative effects of GBA2 were assessed in tumor cells expressing inducible GBA2 and in established melanoma xenografts. As a control an inducible catalytically inactive GBA2 mutant was generated. Sphingolipid levels were monitored by mass spectrometry; unfolded protein response (UPR) and apoptosis were assessed by Western blot and flow cytometry analyses, respectively. We report that GBA2 is down-regulated in melanoma; inducible expression of GBA2 affects endogenous sphingolipid metabolism by promoting glucosylceramide degradation (decrease by 78%) and ceramide generation; this is followed by a UPR that causes apoptosis, subsequent decreased anchorage-independent cell growth, and reduced in vivo tumor growth (by 40%); and all these events are abrogated when expressing a catalytically inactive GBA2. This study documents for the first time the antitumor activity of GBA2 and provides evidence for the role of nonlysosomal glucosylceramide breakdown as a source of bioactive ceramide and a mechanistic link between glycolipid catabolism and the UPR/death response of melanoma cells.

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis.

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.

Genetic disorders of simple sphingolipid metabolism.

A better understanding of the functions sphingolipids play in living organisms can be achieved by analyzing the biochemical and physiological changes that result from genetic alterations of sphingolipid metabolism. This review summarizes the current knowledge gained from studies both on human patients and mutant animals (mice, cats, dogs, and cattle) with genetic disorders of sphingolipid metabolism. Genetic alterations affecting the biosynthesis, transport, or degradation of simple sphingolipids are discussed.

Sphingolipid paracrine signaling impairs keratinocyte adhesion to promote melanoma invasion.

Melanoma is the deadliest form of skin cancer due to its propensity to metastasize. It arises from melanocytes, which are attached to keratinocytes within the basal epidermis. Here, we hypothesize that, in addition to melanocyte-intrinsic modifications, dysregulation of keratinocyte functions could initiate early-stage melanoma cell invasion. We identified the lysolipid sphingosine 1-phosphate (S1P) as a tumor paracrine signal from melanoma cells that modifies the keratinocyte transcriptome and reduces their adhesive properties, leading to tumor invasion. Mechanistically, tumor cell-derived S1P reduced E-cadherin expression in keratinocytes via S1P receptor dependent Snail and Slug activation. All of these effects were blocked by S1P2/3 antagonists. Importantly, we showed that epidermal E-cadherin expression was inversely correlated with the expression of the S1P-producing enzyme in neighboring tumors and the Breslow thickness in patients with early-stage melanoma. These findings support the notion that E-cadherin loss in the epidermis initiates the metastatic cascade in melanoma.

Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine.

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.

The tricyclodecan-9-yl-xanthogenate D609 triggers ceramide increase and enhances FasL-induced caspase-dependent and -independent cell death in T lymphocytes.

D609 is known to modulate death receptor-induced ceramide generation and cell death. We show that in Jurkat cells, non-toxic D609 concentrations inhibit sphingomyelin synthase and, to a lesser extent, glucosylceramide synthase, and transiently increase the intracellular ceramide level. D609 significantly enhanced FasL-induced caspase activation and apoptosis. D609 stimulated FasL-induced cell death in caspase-8-deficient Jurkat cells, indicating that D609 acts downstream of caspase-8. At high FasL concentration (500 ng/mL), cell death was significantly, but not completely, inhibited by zVAD-fmk, a broad-spectrum caspase inhibitor, indicating that FasL can activate both caspase-dependent and -independent cell death signaling pathways. FasL-induced caspase activation was abolished by zVAD-fmk, whereas ceramide production was only partially impaired. D609 enhanced caspase-independent ceramide increase and cell death in response to FasL. Also, D609 overcame zVAD-fmk-conferred resistance to a FasL concentration as low as 50 ng/mL and bypassed RIP deficiency. It is likely that mitochondrial events were involved, since Bcl-xL over-expression impaired D609 effects. In PHA-activated human T lymphocytes, D609 enhanced FasL-induced cell death in the presence or absence of zVAD-fmk. Altogether, our data strongly indicate that the inhibition of ceramide conversion to complex sphingolipids by D609 is accompanied by an enhancement of FasL-induced caspase-dependent and -independent cell death in T lymphocytes.

Potential Role of Sphingolipidoses-Associated Lysosphingolipids in Cancer.

Sphingolipids play a key structural role in cellular membranes and/or act as signaling molecules. Inherited defects of their catabolism lead to lysosomal storage diseases called sphingolipidoses. Although progress has been made toward a better understanding of their pathophysiology, several issues still remain unsolved. In particular, whether lysosphingolipids, the deacylated form of sphingolipids, both of which accumulate in these diseases, are simple biomarkers or play an instrumental role is unclear. In the meanwhile, evidence has been provided for a high risk of developing malignancies in patients affected with Gaucher disease, the most common sphingolipidosis. This article aims at analyzing the potential involvement of lysosphingolipids in cancer. Knowledge about lysosphingolipids in the context of lysosomal storage diseases is summarized. Available data on the nature and prevalence of cancers in patients affected with sphingolipidoses are also reviewed. Then, studies investigating the biological effects of lysosphingolipids toward pro or antitumor pathways are discussed. Finally, original findings exploring the role of glucosylsphingosine in the development of melanoma are presented. While this lysosphingolipid may behave like a protumorigenic agent, further investigations in appropriate models are needed to elucidate the role of these peculiar lipids, not only in sphingolipidoses but also in malignant diseases in general.

Adipocyte Extracellular Vesicles Decrease p16INK4A in Melanoma: An Additional Link between Obesity and Cancer.

Obesity is a recognized factor for increased risk and poor prognosis of many cancers, including melanoma. In this study, using genetically engineered mouse models of melanoma (NrasQ61K transgenic expression, associated or not with Cdkn2a heterozygous deletion), we show that obesity increases melanoma initiation and progression by supporting tumor growth and metastasis, thereby reducing survival. This effect is associated with a decrease in p16INK4A expression in tumors. Mechanistically, adipocytes downregulate p16INK4A in melanoma cells through ß-catenin-dependent regulation, which increases cell motility. Furthermore, ß-catenin is directly transferred from adipocytes to melanoma cells in extracellular vesicles, thus increasing its level and activity, which represses CDKN2A transcription. Adipocytes from individuals with obesity have a stronger effect than those from lean individuals, mainly owing to an increase in the number of vesicles secreted, thus increasing the amount of ß-catenin delivered to melanoma cells and, consequently, amplifying their effect. In conclusion, in this study, we reveal that adipocyte extracellular vesicles control p16INK4A expression in melanoma, which promotes tumor progression. This work expands our understanding of the cooperation between adipocytes and tumors, particularly in obesity.

Asymmetric synthesis and cytotoxic activity of isomeric phytosphingosine derivatives.

New phytosphingosine analogues have been conceived, synthesised and their cytotoxicity in B16 murine melanoma cells tested. These compounds embed an isomeric substitution pattern resulting from a formal permutation of the C-2 and C-4 substituents along the aliphatic skeleton of the original sphingoid base. Five different stereoisomers have been accessed through regio- and stereocontrolled opening of the oxirane of long chain epoxyamine precursors. The corresponding N-hexyl and N-octanoyl derivatives have also been prepared. In cell viability experiments all the primary amines were found to be more active than the natural phytosphingosine with IC(50) in the low µM range for the most potent compounds.

Combining Nivolumab and Ipilimumab with Infliximab or Certolizumab in Patients with Advanced Melanoma: First Results of a Phase Ib Clinical Trial.

PURPOSE: TNF blockers can be used to manage gastrointestinal inflammatory side effects following nivolumab and/or ipilimumab treatment in patients with advanced melanoma. Our preclinical data showed that anti-TNF could promote the efficacy of immune checkpoint inhibitors. PATIENTS AND METHODS: TICIMEL (NTC03293784) is an open-label, two-arm phase Ib clinical trial. Fourteen patients with advanced and/or metastatic melanoma (stage IIIc/IV) were enrolled. Patients were treated with nivolumab (1 mg/kg) and ipilimumab (3 mg/kg) combined to infliximab (5 mg/kg, N = 6) or certolizumab (400/200 mg, N = 8). The primary endpoint was safety and the secondary endpoint was antitumor activity. Adverse events (AEs) were graded according to the NCI Common Terminology Criteria for Adverse Events and response was assessed following RECIST 1.1. RESULTS: Only one dose-limiting toxicity was observed in the infliximab cohort. The two different combinations were found to be safe. We observed lower treatment-related AEs with infliximab as compared with certolizumab. In the certolizumab cohort, one patient was not evaluable for response. In this cohort, four of eight patients exhibited hepatobiliary disorders and seven of seven evaluable patients achieved objective response including four complete responses (CRs) and three partial responses (PRs). In the infliximab cohort, we observed one CR, two PRs, and three progressive diseases. Signs of activation and maturation of systemic T-cell responses were seen in patients from both cohorts. CONCLUSIONS: Our results show that both combinations are safe in human and provide clinical and biological activities. The high response rate in the certolizumab-treated patient cohort deserves further investigations.

Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti-PD-1 Therapy Efficacy.

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1.

Palmitoyl protein thioesterase 1 modulates tumor necrosis factor alpha-induced apoptosis.

Induction of apoptosis by TNF has recently been shown to implicate proteases from lysosomal origin, the cathepsins. Here, we investigated the role in apoptosis of palmitoyl protein thioesterase 1 (PPT1), another lysosomal enzyme that depalmitoylates proteins. We show that transformed fibroblasts derived from patients with the infantile form of neuronal ceroid lipofuscinosis (INCL), a neurodegenerative disease due to deficient activity of PPT1, are partially resistant to TNF-induced cell death (57-75% cell viability vs. 15-30% for control fibroblasts). TNF-initiated proteolytic cleavage of caspase-8, Bid and caspase-3, as well as cytochrome c release was strongly attenuated in INCL fibroblasts as compared to control cells. Noteworthy, activation of p42/p44 mitogen-activated protein kinase and of transcription factor NF-kappaB by TNF, and induction of cell death by staurosporine or chemotherapeutic drugs in INCL cells were unaffected by PPT1 deficiency. Resistance to TNF-induced apoptosis was also observed in embryonic fibroblasts derived from Ppt1/Cln1-deficient mice but not from mice with a targeted deletion of Cln3 or Cln5. Finally, reconstitution of PPT1 activity in mutant cells was accompanied by resensitization to TNF-induced caspase activation and toxicity. These observations emphasize for the first time the role of PPT1 and, likely, protein depalmitoylation in the regulation of TNF-induced apoptosis.

Synthesis of cytotoxic aza analogues of jaspine B.

A straightforward access to pyrrolidine-based analogues of jaspine B was developed. Five stereoisomers were prepared including the all-cis derivatives presenting the configuration of the natural anhydrophytosphingosine. The synthesis of the latter relied on an original Staudinger-type cyclization process. The compounds were evaluated regarding their ability to alter tumor cells' viability and to interfere with the metabolism of sphingolipids.

Flexible and enantioselective access to jaspine B and biologically active chain-modified analogues thereof.

Whereas the all-cis tetrahydrofuran framework of the cytotoxic anhydrophytosphingosine jaspine B is considered as a relevant pharmacophore, little is known about the influence of the aliphatic chain of this amphiphilic molecule on its activity. We developed a synthetic strategy allowing flexible introduction of various lipophilic fragments in the jaspine's skeleton. The route was validated with two distinct approaches to jaspine B. Five chain-modified analogues were also prepared. Biological evaluation of these derivatives demonstrated a good correlation between their cytotoxicity and their capacity to inhibit conversion of ceramide into sphingomyelin in melanoma cells. A series of potent and selective inhibitors of sphingomyelin production was thus identified. Furthermore, the good overall potency of an omega-aminated analogue allowed us to dissociate of the pharmacological action of jaspine B from its amphiphilic nature.

New Insights into the Role of Sphingolipid Metabolism in Melanoma.

Cutaneous melanoma is a deadly skin cancer whose aggressiveness is directly linked to its metastatic potency. Despite remarkable breakthroughs in term of treatments with the emergence of targeted therapy and immunotherapy, the prognosis for metastatic patients remains uncertain mainly because of resistances. Better understanding the mechanisms responsible for melanoma progression is therefore essential to uncover new therapeutic targets. Interestingly, the sphingolipid metabolism is dysregulated in melanoma and is associated with melanoma progression and resistance to treatment. This review summarises the impact of the sphingolipid metabolism on melanoma from the initiation to metastatic dissemination with emphasis on melanoma plasticity, immune responses and resistance to treatments.

Lipid metabolic Reprogramming: Role in Melanoma Progression and Therapeutic Perspectives.

Metabolic reprogramming contributes to the pathogenesis and heterogeneity of melanoma. It is driven both by oncogenic events and the constraints imposed by a nutrient- and oxygen-scarce microenvironment. Among the most prominent metabolic reprogramming features is an increased rate of lipid synthesis. Lipids serve as a source of energy and form the structural foundation of all membranes, but have also emerged as mediators that not only impact classical oncogenic signaling pathways, but also contribute to melanoma progression. Various alterations in fatty acid metabolism have been reported and can contribute to melanoma cell aggressiveness. Elevated expression of the key lipogenic fatty acid synthase is associated with tumor cell invasion and poor prognosis. Fatty acid uptake from the surrounding microenvironment, fatty acid ß-oxidation and storage also appear to play an essential role in tumor cell migration. The aim of this review is (i) to focus on the major alterations affecting lipid storage organelles and lipid metabolism. A particular attention has been paid to glycerophospholipids, sphingolipids, sterols and eicosanoids, (ii) to discuss how these metabolic dysregulations contribute to the phenotype plasticity of melanoma cells and/or melanoma aggressiveness, and (iii) to highlight therapeutic approaches targeting lipid metabolism that could be applicable for melanoma treatment.