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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.

TCGA dataset

This file contains supplementary table 1 and supplementary figures 1-7.

Supplementary Figure S1 shows the effects of PLX on S1PR expression and cell death in BRAFi-sensitive and -resistant melanoma cells

BRAF inhibitors (BRAFi) are used to treat patients with melanoma harboring the V600E mutation. However, resistance to BRAFi is inevitable. Here, we identified sphingosine 1-phosphate (S1P) receptors as regulators of BRAFV600E-mutant melanoma cell-autonomous resistance to BRAFi. Moreover, our results reveal a distinct sphingolipid profile, that is, a tendency for increased very long-chain ceramide species, in the plasma of patients with melanoma who achieve a response to BRAFi therapy as compared with patients with progressive disease. Treatment with BRAFi resulted in a strong decrease in S1PR1/3 expression in sensitive but not in resistant cells. Genetic and pharmacologic interventions, that increase ceramide/S1P ratio, downregulated S1PR expression and blocked BRAFi-resistant melanoma cell growth. This effect was associated with a decreased expression of MITF and Bcl-2. Moreover, the BH3 mimetic ABT-737 improved the antitumor activity of approaches targeting S1P-metabolizing enzymes in BRAFi-resistant melanoma cells. Collectively, our findings indicate that targeting the S1P/S1PR axis could provide effective therapeutic options for patients with melanoma who relapse after BRAFi therapy.

Legends to the supplemental figures

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.

This file contains supplemental tables and figures and their legends.

Supplementary Figure S4. Analysis of the immune response in draining lymph nodes from WT and TNF-deficient mice.

Supplementary information. This file contains supplementary materials and methods, and legends to supplementary figures.

Tissue repair after lesion usually leads to scar healing and thus loss of function in adult mammals. In contrast, other adult vertebrates such as amphibians have the ability to regenerate and restore tissue homeostasis after lesion. Understanding the control of the repair outcome is thus a concerning challenge for regenerative medicine. We recently developed a model of induced tissue regeneration in adult mice allowing the comparison of the early steps of regenerative and scar healing processes. By using studies of gain and loss of function, specific cell depletion approaches, and hematopoietic chimeras we demonstrate here that tissue regeneration in adult mammals depends on an early and transient peak of granulocyte producing reactive oxygen species and an efficient efferocytosis specifically by tissue-resident macrophages. These findings highlight key and early cellular pathways able to drive tissue repair towards regeneration in adult mammals.

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.

BRAF inhibitors (BRAFi) are used to treat patients with melanoma harboring the V600E mutation. However, resistance to BRAFi is inevitable. Here, we identified sphingosine 1-phosphate (S1P) receptors as regulators of BRAFV600E-mutant melanoma cell-autonomous resistance to BRAFi. Moreover, our results reveal a distinct sphingolipid profile, that is, a tendency for increased very long-chain ceramide species, in the plasma of patients with melanoma who achieve a response to BRAFi therapy as compared with patients with progressive disease. Treatment with BRAFi resulted in a strong decrease in S1PR1/3 expression in sensitive but not in resistant cells. Genetic and pharmacologic interventions, that increase ceramide/S1P ratio, downregulated S1PR expression and blocked BRAFi-resistant melanoma cell growth. This effect was associated with a decreased expression of MITF and Bcl-2. Moreover, the BH3 mimetic ABT-737 improved the antitumor activity of approaches targeting S1P-metabolizing enzymes in BRAFi-resistant melanoma cells. Collectively, our findings indicate that targeting the S1P/S1PR axis could provide effective therapeutic options for patients with melanoma who relapse after BRAFi therapy.

Introduction Malgre l’efficacite des anti-PD-1 et anti-CTLA-4 pour le traitement des melanomes avances, plus de 40 % des patients resistent ou rechutent dans les 5 ans suivant l’initiation du traitement et plus de 50 % des patients developpent des effets indesirables inflammatoires de grade 3/4, dont des colites qui peuvent etre traitees par anti-TNFa. Notre equipe a montre que le TNF limite l’efficacite des anti-PD-1 dans des modeles murins de melanome. Materiel et methodes TICIMEL (NTC3293784) est un essai clinique de phase 1b qui a pour objectif d’evaluer la securite de l’association des anti-TNF (infliximab [5 mg/kg] ou certolizumab [400/200 mg]) a la combinaison nivolumab (1 mg/kg)/ipilimumab (3 mg/kg) (ipi3/nivo1) pour le traitement du melanome avance. L’objectif secondaire de TICIMEL est d’evaluer l’activite anti-tumorale. La severite des effets indesirables a ete evaluee selon la terminologie CTCAE et la reponse selon les criteres RECIST 1.1. Resultats Au cours de la premiere partie de cet essai, 6 et 8 patients ont ete respectivement inclus dans les cohortes infliximab et certolizumab. Un seul patient, inclus dans la cohorte infliximab, developpait une DLT (toxicite limitant la dose). La nature des effets indesirables chez les patients des deux cohortes etait similaire a celle decrite precedemment avec ipi3/nivo1. Un nombre plus eleve d’evenements indesirables etait observe chez les patients traites par certolizumab (n = 8) que chez les patients traites par infliximab (n = 6). Sur les 8 patients inclus dans la cohorte certolizumab, 7 etaient evaluables pour la reponse. Tous etaient en reponse objective 12 semaines apres l’initiation du traitement incluant 4 reponses completes (RC). Dans la cohorte infliximab, 3 patients presentaient une reponse objective (1 RC et 2 reponses partielles [RP]) et 3 patients avaient progresse. Enfin, nos analyses exploratoires montrent que les deux tritherapies potentialisent l’activation et la maturation des lymphocytes T dans le sang des patients. Nous observons egalement une augmentation des taux plasmatiques d’IFNg, une cytokine essentielle aux reponses immunitaires anti-tumorales. Discussion Nos premiers resultats montrent que l’association d’anti-TNF a la combinaison ipi3/nivo1 est sure. Nos resultats exploratoires associes au taux eleve de reponse des patients traites par certolizumab suggerent une potentielle synergie entre l’association ipi3/nivo1 et cet anti-TNF. Les cohortes d’expansion de l’essai sont en cours de recrutement.

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.

Simple Summary Melanoma is a devastating skin cancer characterized by an impressive metabolic plasticity. Melanoma cells are able to adapt to the tumor microenvironment by using a variety of fuels that contribute to tumor growth and progression. In this review, the authors summarize the contribution of the lipid metabolic network in melanoma plasticity and aggressiveness, with a particular attention to specific lipid classes such as glycerophospholipids, sphingolipids, sterols and eicosanoids. They also highlight the role of adipose tissue in tumor progression as well as the potential antitumor role of drugs targeting critical steps of lipid metabolic pathways in the context of melanoma. Abstract 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 β-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.

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.

Simple Summary Triple-negative breast cancer (TNBC) is associated with a poor prognosis, and the development of better therapeutic strategies is required. Herein, we investigated the role of the anti-angiogenic thrombospondin-1 (TSP1) in TNBC. TSP1 expression in tumor biopsies from TNBC patients was associated with a bad prognosis and a weak content of tumor-infiltrating lymphocytes (TILs). In the 4T1 mouse TNBC model, TSP1 knockdown reduced TGF-β activation and enhanced the content of TILs. Moreover, TSP1 knockdown decreased lung metastasis in syngeneic Balb/c immunocompetent mice but not in immunodeficient nude mice. Finally, TSP1 knockdown enhanced anti-PD-1 immunotherapy efficacy. Thus, targeting TSP1 may be considered as a putative therapeutic strategy in TNBC in combination with immunotherapy. Abstract Triple-negative breast cancer (TNBC) is notoriously aggressive with a high metastatic potential, and targeted therapies are lacking. Using transcriptomic and histologic analysis of TNBC samples, we found that a high expression of thrombospondin-1 (TSP1), a potent endogenous inhibitor of angiogenesis and an activator of latent transforming growth factor beta (TGF-β), is associated with (i) gene signatures of epithelial–mesenchymal transition and TGF-β signaling, (ii) metastasis and (iii) a reduced survival in TNBC patients. In contrast, in tumors expressing low levels of TSP1, gene signatures of interferon gamma (IFN-γ) signaling and lymphocyte activation were enriched. In TNBC biopsies, TSP1 expression inversely correlated with the CD8+ tumor-infiltrating lymphocytes (TILs) content. In the 4T1 metastatic mouse model of TNBC, TSP1 silencing did not affect primary tumor development but, strikingly, impaired metastasis in immunocompetent but not in immunodeficient nude mice. Moreover, TSP1 knockdown increased tumor vascularization and T lymphocyte infiltration and decreased TGF-β activation in immunocompetent mice. Noteworthy was the finding that TSP1 knockdown increased CD8+ TILs and their programmed cell death 1 (PD-1) expression and sensitized 4T1 tumors to anti-PD-1 therapy. TSP1 inhibition might thus represent an innovative targeted approach to impair TGF-β activation and breast cancer cell metastasis and improve lymphocyte infiltration in tumors, and immunotherapy efficacy in TNBC.

Macrophage-mediated cytotoxicity is controlled by surface receptor expression and activation. Despite the numerous studies documenting the role of macrophage C-type lectin receptors (CLR) in pathogen elimination, little is known about their contribution to antitumor responses. Here, we report that IL13 inhibits T-cell lymphoma and ovarian adenocarcinoma development in tumor-bearing mice through the conversion of tumor-supporting macrophages to cytotoxic effectors, characterized by a CLR signature composed of dectin-1 and mannose receptor (MR). We show that dectin-1 and MR are critical for the recognition of tumor cells through sialic acid–specific glycan structure on their surface and for the subsequent activation of macrophage tumoricidal response. Finally, we validated that IL13 antitumor effect mediated by dectin-1 and MR overexpression on macrophages can extend to various types of human tumors. Therefore, these results identify these CLRs as potential targets to promote macrophage antitumor response and represent an attractive approach to elicit tumor-associated macrophage tumoricidal properties.

Sphingolipids represent a major class of lipids that are essential constituents of eukaryotic cells. They are predominantly located in plasma membrane microdomains, and play an important structural role in regulating membrane fluidity. They are also bioactive effectors involved in diverse key cellular functions such as apoptosis and proliferation. The implication of some sphingolipids in cancer is well established whereas that of some others is still a matter of intense investigation. Glucosylceramide is the backbone of more than 300 structurally different glycosphingolipids including gangliosides and sulfatides, and is essential for mammalian development. Therefore, glucosylceramidases (also named GBA1, GBA2 and GBA3 β-glucosidases), the enzymes that hydrolyse β-glucosylceramide, play important functions. GBA1 is a lysosomal hydrolase whose deficiency causes Gaucher disease, the most prevalent inherited lysosomal storage disorder. GBA2 is a ubiquitous non-lysosomal glucosylceramidase whose mutations have been associated with some forms of hereditary spastic paraplegia. GBA3 is a cytosolic β-glucosidase, mostly present in the kidney, liver, spleen, intestine and lymphocytes of mammals, the function of which is still unclear. Whereas glucosylceramide synthase is implicated in multidrug resistance, the role of glucosylceramide breakdown in cancer is not yet fully appreciated. Defective GBA1 enzyme activity in humans, i.e., Gaucher disease, is associated with an increased risk of multiple myeloma and other malignancies. Putative molecular links between Gaucher disease and cancer, which might implicate the malignant cell and/or its microenvironment, are reviewed. The functions of GBA2 and GBA3 in cancer progression are also discussed.

During their life, T cells are constantly circulating throughout the body. Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, triggers T cell egress from the thymus and secondary lymphoid organs (SLO) into the lymph and the blood. This phenomenon is dependent on the S1P gradient between lymphoid organs (including thymus and SLO) and the lymphatic and blood vessels, which exhibit low, intermediate and high S1P levels, respectively. T cells follow the S1P gradient via the engagement of the G-protein-coupled receptor S1P receptor 1 (S1P1) expressed on T cells.1 S1P production is regulated by various sphingolipid-metabolizing enzymes. The sphingosine kinases 1 and 2 (SKs), encoded by Sphk1 and Sphk2, phosphorylate the sphingosine (Sph) into S1P. Conversely, S1P can be dephosphorylated to Sph by S1P phosphatases 1 and 2, encoded by Sgpp1 and Sgpp2. Alternatively, S1P can be irreversibly degraded by the S1P Lyase (SPL), encoded by Sgpl1, into phosphoethanolamine and hexadecenal. The secretion of S1P was described to involve two transporters, the ATP-binding cassette (ABC) transporter, in a non-specific manner, and the recently identified specific transporter Spinster 2 (SPNS2), encoded by the Spns2 gene.2 As a result, Spns2-deficient mice exhibit decreased concentrations of S1P into the lymph but not blood.3 In a recent study, Mendoza et al. reported that S1P, which is secreted by lymphatic endothelial cells via SPNS2, favors naive T-cell survival by maintaining their mitochondrial content in an S1P1-dependent manner.4 Herein, we discuss the main findings and pathophysiological implications of the latter study as regard to the state of the art.

Sphingolipids are sphingoid base-containing lipids, among which some metabolites behave as bioactive molecules in various biological processes, including cell death. Whereas ceramide is now viewed as an anti-oncometabolite, leading to cancer cell death, CD95L-induced apoptosis is associated with sphingolipid changes, which likely contribute to caspase-dependent signaling pathway activation. Here, we describe Liquid Chromatography-high resolution mass spectrometry method (LC-HRMS) to analyze sphingolipid metabolism changes triggered by CD95L.

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.

Sphingolipids are sphingoid base-containing lipids, among which some metabolites behave as bioactive molecules in various biological processes, including cell death. Whereas ceramide is now viewed as an anti-oncometabolite, leading to cancer cell death, CD95L-induced apoptosis is associated with sphingolipid changes, which likely contribute to caspase-dependent signaling pathway activation. Here, we describe Liquid Chromatography-high resolution mass spectrometry method (LC-HRMS) to analyze sphingolipid metabolism changes triggered by CD95L.

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.

Sphingolipids (SLs) are sphingoid base-containing lipids, which are enriched in plasma membrane microdomains. Some SLs behave as bioactive molecules, modulating cell signalling in various pathophysiological contexts. Whereas ceramide triggers apoptosis and impairs cell migration, sphingosine-1-phosphate (S1P) induces the opposite effects. CD95/Fas, TRAIL-R1 (DR4), and TRAIL-R2 (DR5) are death receptors (DRs) and members of the TNF-R1 superfamily. DRs trigger apoptosis of various cell types, including cancer cells. Over the last two decades, a growing body of evidence indicates that SLs modulate DR signalling. DR stimulation triggers the generation of SLs, including ceramide, sphingosine, and gangliosides. Ceramide has been reported to facilitate DR clustering into lipid rafts upon pro-apoptotic DR agonists. Moreover, ceramide and its metabolites likely contribute to the mitochondrial route of apoptosis. More recently, SLs have been shown to modulate CD95-mediated cell migration of triple negative breast cancer cell lines and Th17 lymphoid cells in response to a nonapoptotic form of CD95L. Herein, we review the role of SLs in DR signalling, including apoptotic and migration pathways.

International audience; Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.Oncogene advance online publication, 17 March 2014; doi:10.1038/onc.2014.55.

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

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.

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.

Porphyrins are used as photosensitizer (PS) in photodynamic therapy in cancer treatment. Nevertheless, the development of photochemotherapy in oncology remains limited, because of the low selectivity of PSs. In order to allow PS targeting toward tumor-associated antigens, for the first time a white-light activatable porphyrin, [5-(4-(5-carboxy-1-butoxy)-phenyl)-10,15,20-tris(4-N-methyl)-pyridiniumyl)-porphyrin] (TrMPyP) was covalently linked to Morniga G (MorG), a galactose-specific binding plant lectin, known to recognize with high-affinity tumor-associated T/Tn antigen in cell-free systems. Firstly, using fluorescein-labeled MorG, the sugar-dependent binding and uptake of lectin by Tn-positive (Jurkat lymphoid leukemia) cells was demonstrated. Secondly, the TrMPyP-MorG conjugate was molecularly characterized. Cytometric and confocal microscopic analysis demonstrated that PS covalent linking to MorG preserved sugar-dependent specific binding and uptake of lectin by Jurkat leukemia lymphocytes. Thirdly, the conjugate (with a 1:1 PS:lectin ratio) that was bound and quickly (5 min) taken-up, induced greater than 90% cytotoxicity upon irradiation at 10 nm concentration, whereas the free PS was absolutely nontoxic. On the contrary, normal lymphocytes strongly resisted to the conjugate-mediated phototoxicity. Thus, owing to their binding and endocytosis capacities, plant lectins represent promising molecules for targeting of tumor glycan alteration and to enhance the efficiency of specific delivery of PSs to tumor cells.

In a recent patent, Wanebo HJ proposed to increase "apoptosis in a cancer cell comprising contacting the cancer cell with (a) oxaliplatin and (b) C6-ceramide, sequentially or concomitantly". Cisplatin and derivatives are commonly used in the treatment of solid tumors. Cisplatin induces DNA adducts, which are responsible for the induction of a cellular stress, leading to the elimination of the proliferating cells by apoptosis. Recently, a different mechanism has been reported to explain the tumoricidal action of platinum-based agents. Indeed, cells treated with cisplatin exhibit an increase in plasma membrane fluidity through the activation of acid sphingomyelinase, the subsequent generation of ceramide and the redistribution of the death receptor CD95 into the lipid rafts. This latter event promotes the initiation of the apoptotic signal and the elimination of the malignant cells. In this review, we discuss the potential role played by the death receptor in the potentiation effect of exogenously added ceramide upon the oxaliplatin-mediated cytotoxic effect.

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-beta1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-beta1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth. Mrad, Marguerite Imbert, Caroline Garcia, Virginie Rambow, Florian Therville, Nicole Carpentier, Stephane Segui, Bruno Levade, Thierry Azar, Rania Marine, Jean-Christophe Diab-Assaf, Mona Colacios, Celine Andrieu-Abadie, Nathalie eng 2016/10/07 06:00 Oncotarget. 2016 Nov 1;7(44):71873-71886. doi: 10.18632/oncotarget.12380. The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-beta1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-beta1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth. ispartof: Oncotarget vol:7 issue:44 pages:71873-71886 ispartof: location:United States status: published