27-Hydroxylation of oncosterone by CYP27A1 switches its activity from pro-tumor to anti-tumor.

Oncosterone (6-oxo-cholestane-3ß,5α-diol; OCDO) is an oncometabolite and a tumor promoter on estrogen receptor alpha-positive breast cancer (ER(+) BC) and triple-negative breast cancers (TN BC). OCDO is an oxysterol formed in three steps from cholesterol: 1) oxygen addition at the double bond to give α- or ß- isomers of 5,6-epoxycholestanols (5,6-EC), 2) hydrolyses of the epoxide ring of 5,6-ECs to give cholestane-3ß,5α,6ß-triol (CT), and 3) oxidation of the C6 hydroxyl of CT to give OCDO. On the other hand, cholesterol can be hydroxylated by CYP27A1 at the ultimate methyl carbon of its side chain to give 27-hydroxycholesterol ((25R)-Cholest-5-ene-3beta,26-diol, 27HC), which is a tumor promoter for ER(+) BC. It is currently unknown whether OCDO and its precursors can be hydroxylated at position C27 by CYP27A1, as is the impact of such modification on the proliferation of ER(+) and TN BC cells. We investigated, herein, whether 27H-5,6-ECs ((25R)-5,6-epoxycholestan-3ß,26-diol), 27H-CT ((25R)-cholestane-3ß,5α,6ß,26-tetrol) and 27H-OCDO ((25R)-cholestane-6-oxo-3ß,5α,26-triol) exist as metabolites and can be produced by cells expressing CYP27A1. We report, for the first time, that these compounds exist as metabolites in humans. We give pharmacological and genetic evidence that CYP27A1 is responsible for their production. Importantly, we found that 27-hydroxy-OCDO (27H-OCDO) inhibits BC cell proliferation and blocks OCDO and 27-HC-induced proliferation in BC cells, showing that this metabolic conversion commutes the proliferative properties of OCDO into antiproliferative ones. These data suggest an unprecedented role of CYP27A1 in the control of breast carcinogenesis by inhibiting the tumor promoter activities of oncosterone and 27-HC.

Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor.

Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3ß,5α,6ß-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) by 11ß-hydroxysteroid-dehydrogenase-type-2 (11ßHSD2). 11ßHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11ßHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11ßHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11ßHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.

Natural and semisynthetic oxyprenylated aromatic compounds as stimulators or inhibitors of melanogenesis.

It has been very recently shown how naturally occurring oxyprenylated coumarins are effective modulators of melanogenesis. In this short communication we wish to generalize the potentialities as skin tanning or whitening agents of a wider panel of natural and semisynthetic aromatic compounds, including coumarins, cinnamic and benzoic acids, cinnamaldehydes, benzaldehyde, and anthraquinone derivatives. A total number of 43 compounds have been tested assaying their capacity to inhibit or stimulate melanin biosynthesis in cultured murine Melan A cells. The wider number of chemicals herein under investigation allowed to depict a detailed structure-activity relationship, as the following: (a) benzoic acid derivatives are slightly pigmenting agent, for which the effect is more pronounced in compounds with longer O-side chains; (b) independently from the type of substitution, cinnamic acids are able to increase melanin biosynthesis, while benzaldehydes are able to decrease it; (c) coumarins with a 3,3-dimethylallyl or shorter skeletons as substituents in position 7 are tanning agents, while coumarins with farnesyloxy groups are whitening ones; (d) double oxyprenylation in position 6 and 7 and 3,3-dimethylallyl or geranyl skeletons have slight depigmenting capacities, while farnesyl skeletons tend to marginally increase the tanning effect; (e) the presence of electron withdrawing groups (acetyl, COOH, and -Cl) and geranyl or farnesyl oxyprenylated chains respectively in positions 3 and 7 of the coumarin nucleus lead to a whitening effect, and finally (f) oxyprenylated anthraquinones have only a weak depigmenting capacity.

HPLC Analysis and Skin Whitening Effects of Umbelliprenin-containing Extracts of Anethum Graveolens, Pimpinella Anisum, and Ferulago Campestris.

Umbelliprenin has recently been shown to have great potential as a skin whitening agent. Wishing to investigate the same effect in plant species known to biosynthesize this coumarin, three plants belonging to the Apiaceae family, namely Anethum graveolens L. (dill), Pimpinella anisum L. (anise), and Ferulago campestris (Besser) Grecescu (field ferula) were screened by HPLC analysis for their respective content of umbelliprenin in extracts obtained with different solvent mixtures and by maceration and ultrasound-assisted processes. EtOH was shown to be the best solvent, providing umbelliprenin yields ranging from 1.7% to 14.4% (with respect to the total amount of extract obtained). Extracts with the highest content of this farnesyloxycoumarin were then assayed as modulators of melanogenesis in cultured murine Melan A cells employing the same umbelliprenin obtained by chemical synthesis as the reference. A parallelism between the content of the coumarin and the recorded depigmenting effect (60% for the EtOH extract of F. campestris as the best value) was revealed for all plants extracts when applied at a dose of 100 µg/mL. Our results demonstrate that the same potential of umbelliprenin can be ascribed also to umbelliprenin-enriched plant extracts which reinforces enforce the widespread use of phyto-preparations for cosmetic purposes (e.g., A. graveolens).

Extracellular vesicles: lipids as key components of their biogenesis and functions.

Intercellular communication has been known for decades to involve either direct contact between cells or to operate via circulating molecules, such as cytokines, growth factors, or lipid mediators. During the last decade, we have begun to appreciate the increasing importance of intercellular communication mediated by extracellular vesicles released by viable cells either from plasma membrane shedding (microvesicles, also named microparticles) or from an intracellular compartment (exosomes). Exosomes and microvesicles circulate in all biological fluids and can trigger biological responses at a distance. Their effects include a large variety of biological processes, such as immune surveillance, modification of tumor microenvironment, or regulation of inflammation. Extracellular vesicles can carry a large array of active molecules, including lipid mediators, such as eicosanoids, proteins, and nucleic acids, able to modify the phenotype of receiving cells. This review will highlight the role of the various lipidic pathways involved in the biogenesis and functions of microvesicles and exosomes.

When cholesterol meets histamine, it gives rise to dendrogenin A: a tumour suppressor metabolite.

Dendrogenin A (DDA) is the first steroidal alkaloid (SA) to be identified in human tissues to date and arises from the stereoselective enzymatic conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA). DDA induces the re-differentiation of cancer cellsin vitroandin vivoand prevents breast cancer (BC) and melanoma development in mice, evidencing its protective role against oncogenesis. In addition, DDA production is lower in BCs compared with normal tissues, suggesting a deregulation of its biosynthesis during carcinogenesis. The discovery of DDA reveals the existence of a new metabolic pathway in mammals which lies at the crossroads of cholesterol and HA metabolism and which leads to the production of this metabolic tumour suppressor.

Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies.

Exosomes are nanovesicles that have emerged as a new intercellular communication system between an intracellular compartment of a donor cell towards the periphery or an internal compartment of a recipient cell. The bioactivity of exosomes resides not only in their protein and RNA contents but also in their lipidic molecules. Exosomes display original lipids organized in a bilayer membrane and along with the lipid carriers such as fatty acid binding proteins that they contain, exosomes transport bioactive lipids. Exosomes can vectorize lipids such as eicosanoids, fatty acids, and cholesterol, and their lipid composition can be modified by in-vitro manipulation. They also contain lipid related enzymes so that they can constitute an autonomous unit of production of various bioactive lipids. Exosomes can circulate between proximal or distal cells and their fate can be regulated in part by lipidic molecules. Compared to their parental cells, exosomes are enriched in cholesterol and sphingomyelin and their accumulation in cells might modulate recipient cell homeostasis. Exosome release from cells appears to be a general biological process. They have been reported in all biological fluids from which they can be recovered and can be monitors of specific pathophysiological situations. Thus, the lipid content of circulating exosomes could be useful biomarkers of lipid related diseases. Since the first lipid analysis of exosomes ten years ago detailed knowledge of exosomal lipids has accumulated. The role of lipids in exosome fate and bioactivity and how they constitute an additional lipid transport system are considered in this review.

One step synthesis of 6-oxo-cholestan-3ß,5α-diol.

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3ß,5α,6ß-triol (CT) and its putative metabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [(14)C]-cholestane-3ß,5α,6ß-triol and [(14)C]-6-oxo-cholestan-3ß,5α-diol. In the present study we report the one-step synthesis of [(14)C]-cholestane-3ß,5α,6ß-triol and [(14)C]-6-oxo-cholestan-3ß,5α-diol by oxidation of [(14)C]-cholesterol with iodide metaperiodate (HIO4).

The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells.

Dendrogenin A (DDA) and dendrogenin B (DDB) are new aminoalkyl oxysterols which display re-differentiation of tumor cells of neuronal origin at nanomolar concentrations. We analyzed the influence of dendrogenins on adult mice neural stem cell proliferation, sphere formation and differentiation. DDA and DDB were found to have potent proliferative effects in neural stem cells. Additionally, they induce neuronal outgrowth from neurospheres during in vitro cultivation. Taken together, our results demonstrate a novel role for dendrogenins A and B in neural stem cell proliferation and differentiation which further increases their likely importance to compensate for neuronal cell loss in the brain.

Identification and pharmacological characterization of cholesterol-5,6-epoxide hydrolase as a target for tamoxifen and AEBS ligands.

The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.

Targeting NR1H/liver X receptor with dendrogenin A differentiates tumor cells to activate a new secretory pathway releasing immunogenic anti-tumor vesicles enriched in LC3-II-associated exosomes.

Normal cells secrete small extracellular vesicles (sEV), containing exosomes and/or ectosomes, which play a beneficial role in monitoring tissue integrity and immune response, whereas cancer cells constitutively secrete sEV, which contribute to inhibit the immune defenses and promote tumor progression and aggressiveness. Therefore, there is a great interest in reprograming tumor sEV functions toward normal ones. We hypothesized that this could be realized by inducing tumor cell re-differentiation with dendrogenin A (DDA), an endogenous oxysterol and a ligand of NR1 H/LXR (nuclear receptor subfamily 1 group H). At low doses, DDA induces tumor cell differentiation, tumor growth inhibition and immune cell infiltration into tumors. At high doses, DDA induces lethal macroautophagy/autophagy in tumors by increasing LC3 expression at the mRNA and protein level, through NR1H2/LXRß. In the present study, we showed that low doses of DDA re-differentiate tumor cells by interacting with NR1H2. This results in an increased formation of multivesicular bodies (MVB) in tumor cells and an enhanced secretion of LC3-II-associated exosome-enriched sEV, with immune and anticancer properties. This study highlights the original LC3-II-associated exosome secretory pathway driven by the DDA-NR1H2 complex and paves the way to the development of new therapeutic strategies against pro-tumor exosomes.

Is cholesterol a risk factor for breast cancer incidence and outcome?

Cholesterol plays important roles in many physiological processes, including cell membrane structure and function, hormone synthesis, and the regulation of cellular homeostasis. The role of cholesterol in breast cancer is complex, and some studies have suggested that elevated cholesterol levels may be associated with an increased risk of developing breast cancer, while others have found no significant association. On the other hand, other studies have shown that, for total cholesterol and plasma HDL-associated cholesterol levels, there was inverse association with breast cancer risk. One possible mechanism by which cholesterol may contribute to breast cancer risk is as a key precursor of estrogen. Other potential mechanisms by which cholesterol may contribute to breast cancer risk include its role in inflammation and oxidative stress, which have been linked to cancer progression. Cholesterol has also been shown to play a role in signaling pathways regulating the growth and proliferation of cancer cells. In addition, recent studies have shown that cholesterol metabolism can generate tumor promoters such as cholesteryl esters, oncosterone, 27-hydroxycholesterol but also tumor suppressor metabolites such as dendrogenin A. This review summarizes some of the most important clinical studies that have evaluated the role of cholesterol or its derivatives in breast cancer. It also addresses the role of cholesterol and its derivatives at the cellular level.

Chemical synthesis and biochemical properties of cholestane-5α,6ß-diol-3-sulfonate: A non-hydrolysable analogue of cholestane-5α,6ß-diol-3ß-sulfate.

Cholestane-3ß,5α,6ß-triol (CT) is a primary metabolite of 5,6-epoxycholesterols (5,6-EC) that is catalyzed by the cholesterol-5,6-epoxide hydrolase (ChEH). CT is a well-known biomarker for Niemann-Pick disease type C (NP-C), a progressive inherited neurodegenerative disease. On the other hand, CT is known to be metabolized by the 11ß-hydroxysteroid-dehydrogenase of type 2 (11ß-HSD2) into a tumor promoter named oncosterone that stimulates the growth of breast cancer tumors. Sulfation is a major metabolic transformation leading to the production of sulfated oxysterols. The production of cholestane-5α,6ß-diol-3ß-O-sulfate (CDS) has been reported in breast cancer cells. However, no data related to CDS biological properties have been reported so far. These studies have been hampered because sulfate esters of sterols and steroids are rapidly hydrolyzed by steroid sulfatase to give free steroids and sterols. In order to get insight into the biological properties of CDS, we report herein the synthesis and the characterization of cholestane-5α,6ß-diol-3ß-sulfonate (CDSN), a non-hydrolysable analogue of CDS. We show that CDSN is a potent inhibitor of 11ß-HSD2 that blocks oncosterone production on cell lysate. The inhibition of oncosterone biosynthesis of a whole cell assay was observed but results from the blockage by CDSN of the uptake of CT in MCF-7 cells. While CDSN inhibits MCF-7 cell proliferation, we found that it potentiates the cytotoxic activity of post-lanosterol cholesterol biosynthesis inhibitors such as tamoxifen and PBPE. This effect was associated with an increase of free sterols accumulation and the appearance of giant multilamellar bodies, a structural feature reminiscent of Type C Niemann-Pick disease cells and consistent with a possible inhibition by CDSN of NPC1. Altogether, our data showed that CDSN is biologically active and that it is a valuable tool to study the biological properties of CDS and more specifically its impact on immunity and viral infection.

An Update on Tamoxifen and the Chemo-Preventive Potential of Vitamin E in Breast Cancer Management.

Breast cancer (BC) is the most common female cancer in terms of incidence and mortality worldwide. Tamoxifen (Nolvadex) is a widely prescribed, oral anti-estrogen drug for the hormonal treatment of estrogen-receptor-positive BC, which represents 70% of all BC subtypes. This review assesses the current knowledge on the molecular pharmacology of tamoxifen in terms of its anticancer and chemo-preventive actions. Due to the importance of vitamin E compounds, which are widely taken as a supplementary dietary component, the review focuses only on the potential importance of vitamin E in BC chemo-prevention. The chemo-preventive and onco-protective effects of tamoxifen combined with the potential effects of vitamin E can alter the anticancer actions of tamoxifen. Therefore, methods involving an individually designed, nutritional intervention for patients with BC warrant further consideration. These data are of great importance for tamoxifen chemo-prevention strategies in future epidemiological studies.

Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles.

We recently established that drugs used for the treatment and the prophylaxis of breast cancers, such as tamoxifen, were potent inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), which led to the accumulation of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6ß-epoxy-cholesterol (5,6ß-EC). This could be considered a paradox because epoxides are known as alkylating agents with putative carcinogenic properties. We report here that, as opposed to the carcinogen styrene-oxide, neither of the ECs reacted spontaneously with nucleophiles. Under catalytic conditions, 5,6ß-EC remains unreactive whereas 5,6α-EC gives cholestan-3ß,5α-diol-6ß-substituted compounds. These data showed that 5,6-ECs are stable epoxides and unreactive toward nucleophiles in the absence of a catalyst, which contrasts with the well-known reactivity of aromatic and aliphatic epoxides. These data rule out 5,6-EC acting as spontaneous alkylating agents. In addition, these data support the existence of a stereoselective metabolism of 5,6α-EC.

Oxysterols are potential physiological regulators of ageing.

Delaying and even reversing ageing is a major public health challenge with a tremendous potential to postpone a plethora of diseases including cancer, metabolic syndromes and neurodegenerative disorders. A better understanding of ageing as well as the development of innovative anti-ageing strategies are therefore an increasingly important field of research. Several biological processes including inflammation, proteostasis, epigenetic, oxidative stress, stem cell exhaustion, senescence and stress adaptive response have been reported for their key role in ageing. In this review, we describe the relationships that have been established between cholesterol homeostasis, in particular at the level of oxysterols, and ageing. Initially considered as harmful pro-inflammatory and cytotoxic metabolites, oxysterols are currently emerging as an expanding family of fine regulators of various biological processes involved in ageing. Indeed, depending of their chemical structure and their concentration, oxysterols exhibit deleterious or beneficial effects on inflammation, oxidative stress and cell survival. In addition, stem cell differentiation, epigenetics, cellular senescence and proteostasis are also modulated by oxysterols. Altogether, these data support the fact that ageing is influenced by an oxysterol profile. Further studies are thus required to explore more deeply the impact of the "oxysterome" on ageing and therefore this cholesterol metabolic pathway constitutes a promising target for future anti-ageing interventions.

Targeting the liver X receptor with dendrogenin A differentiates tumour cells to secrete immunogenic exosome-enriched vesicles.

Tumour cells are characterized by having lost their differentiation state. They constitutively secrete small extracellular vesicles (sEV) called exosomes when they come from late endosomes. Dendrogenin A (DDA) is an endogenous tumour suppressor cholesterol-derived metabolite. It is a new class of ligand of the nuclear Liver X receptors (LXR) which regulate cholesterol homeostasis and immunity. We hypothesized that DDA, which induces tumour cell differentiation, inhibition of tumour growth and immune cell infiltration into tumours, could functionally modify sEV secreted by tumour cells. Here, we have shown that DDA differentiates tumour cells by acting on the LXRß. This results in an increased production of sEV (DDA-sEV) which includes exosomes. The DDA-sEV secreted from DDA-treated cells were characterized for their content and activity in comparison to sEV secreted from control cells (C-sEV). DDA-sEV were enriched, relatively to C-sEV, in several proteins and lipids such as differentiation antigens, "eat-me" signals, lipidated LC3 and the endosomal phospholipid bis(monoacylglycero)phosphate, which stimulates dendritic cell maturation and a Th1 T lymphocyte polarization. Moreover, DDA-sEV inhibited the growth of tumours implanted into immunocompetent mice compared to control conditions. This study reveals a pharmacological control through a nuclear receptor of exosome-enriched tumour sEV secretion, composition and immune function. Targeting the LXR may be a novel way to reprogram tumour cells and sEV to stimulate immunity against cancer.

The 5,6-epoxycholesterol metabolic pathway in breast cancer: Emergence of new pharmacological targets.

Metabolic pathways have emerged as cornerstones in carcinogenic deregulation providing new therapeutic strategies for cancer management. Recently, a new branch of cholesterol metabolism has been discovered involving the biochemical transformation of 5,6-epoxycholesterols (5,6-ECs). The 5,6-ECs are metabolized in breast cancers to the tumour promoter oncosterone whereas, in normal breast tissue, they are metabolized to the tumour suppressor metabolite, dendrogenin A (DDA). Blocking the mitogenic and invasive potential of oncosterone will present new opportunities for breast cancer treatment. The reactivation of DDA biosynthesis, or its use as a drug, represents promising therapeutic approaches such as DDA-deficiency complementation, activation of breast cancer cell re-differentiation and breast cancer chemoprevention. This review presents current knowledge of the 5,6-EC metabolic pathway in breast cancer, focusing on the 5,6-EC metabolic enzymes ChEH and HSD11B2 and on 5,6-EC metabolite targets, the oxysterol receptor (LXRß) and the glucocorticoid receptor. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

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.

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins.

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.