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Introduction: Advanced cutaneous melanoma is a skin cancer characterized by a poor prognosis and high metastatic potential. During metastatic spread, melanoma cells often undergo dedifferentiation toward an invasive phenotype, resulting in reduced expression of microphthalmia-associated transcription factor (MITF)-dependent melanoma antigens and facilitating immune escape. Tumor Necrosis Factor (TNF) is known to be a key factor in melanoma dedifferentiation. Interestingly, accumulating evidence suggests that TNF may play a role in melanoma progression and resistance to immunotherapies. Additionally, TNF has been identified as a potent regulator of sphingolipid metabolism, which could contribute to melanoma aggressiveness and the process of melanoma dedifferentiation. Methods: We conducted RNA sequencing and mass spectrometry analyses to investigate TNF-induced dedifferentiation in two melanoma cell lines. In vitro experiments were performed to manipulate sphingolipid metabolism using genetic or pharmacologic alterations in combination with TNF treatment, aiming to elucidate the potential involvement of this metabolism in TNF-induced dedifferentiation. Lastly, to evaluate the clinical significance of our findings, we performed unsupervised analysis of plasma sphingolipid levels in 48 patients receiving treatment with immune checkpoint inhibitors, either alone or in combination with anti-TNF therapy. Results: Herein, we demonstrate that TNF-induced melanoma cell dedifferentiation is associated with a global modulation of sphingolipid metabolism. Specifically, TNF decreases the expression and activity of acid ceramidase (AC), encoded by the ASAH1 gene, while increasing the expression of glucosylceramide synthase (GCS), encoded by the UGCG gene. Remarkably, knockdown of AC alone via RNA interference is enough to induce melanoma cell dedifferentiation. Furthermore, treatment with Eliglustat, a GCS inhibitor, inhibits TNF-induced melanoma cell dedifferentiation. Lastly, analysis of plasma samples from patients treated with immune checkpoint inhibitors, with or without anti-TNF therapy, revealed significant predictive sphingolipids. Notably, the top 8 predictive sphingolipids, including glycosphingolipids, were associated with a poor response to immunotherapy. Discussion: Our study highlights that ceramide metabolism alterations are causally involved in TNF-induced melanoma cell dedifferentiation and suggests that the evolution of specific ceramide metabolites in plasma may be considered as predictive biomarkers of resistance to immunotherapy.
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Desdiferenciación Celular , Ceramidas , Resistencia a Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Melanoma , Factor de Necrosis Tumoral alfa , Humanos , Melanoma/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Ceramidas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular Tumoral , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/inmunología , Masculino , Glucosiltransferasas/metabolismo , Glucosiltransferasas/genética , Esfingolípidos/metabolismo , Ceramidasa Ácida/metabolismo , Ceramidasa Ácida/genética , Femenino , Persona de Mediana Edad , AncianoRESUMEN
A concise asymmetric synthesis of clickable enantiomeric pyrrolidines was achieved using Crabbé-Ma allenation. The synthesized iminosugars were grafted by copper-free strain-promoted alkyne-azide cycloaddition onto phosphorus dendrimers. The hexavalent and dodecavalent pyrrolidines were evaluated as ß-glucocerebrosidase inhibitors. The level of inhibition suggests that monofluorocyclooctatriazole group may contribute to the affinity for the protein leading to potent multivalent inhibitors. Docking studies were carried out to rationalize these results. Then, the iminosugars clusters were evaluated as pharmacological chaperones in Gaucher patients' fibroblasts. An increase in ß-glucocerebrosidase activity was observed with hexavalent and dodecavalent pyrrolidines at concentrations as low as 1 µM and 0.1 µM, respectively. These iminosugar clusters constitute the first example of multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease.
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Enfermedad de Gaucher , Iminoazúcares , Humanos , Enfermedad de Gaucher/tratamiento farmacológico , Glucosilceramidasa , Pirrolidinas/farmacología , Inhibidores Enzimáticos/farmacologíaRESUMEN
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.
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Melanoma , Humanos , Melanoma/patología , Esfingolípidos/metabolismo , Comunicación Paracrina , Queratinocitos/metabolismo , Cadherinas/metabolismo , Esfingosina/metabolismo , Lisofosfolípidos/metabolismoRESUMEN
MAIN CONCLUSION: After bud burst, a transcriptional reprogramming of the shikimate and phenylpropanoid pathways occurs in grapevine canes resulting in the accumulation of stilbenoids like resveratrol and viniferin. Stilbenoids are phenylpropanoid compounds with important biological properties and biotechnological applications that are synthesized in grapevine in response to different stresses. Although they are found in woody tissues, such as canes and buds, their biosynthesis and accumulation have been essentially described in berries. We have previously shown that transcripts encoding secondary metabolism enzymes accumulate in grapevine canes following the transition from dormancy (E-L 1) to bud burst (E-L 4) suggesting that secondary metabolites may accumulate in grapevine canes during this transition. In the present study, using UPLC-MS we demonstrate the accumulation of important metabolites such as ferulic acid and the stilbenoids E-resveratrol, E-piceatannol and E-ε-viniferin. Stilbenoids accumulation correlated with the increased expression of several stilbene synthase genes and of VviMYB14, encoding a transcription factor that regulates stilbene biosynthesis. In addition, a general stimulation of the plastidial shikimate pathway was observed. Taken together, results show that important secondary metabolites accumulate in the woody canes during bud burst. These findings may aid biotechnological approaches aimed at extracting biologically active phenolic compounds, including stilbenoids, from grapevine woody tissues.
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Espectrometría de Masas en Tándem , Madera , Cromatografía Liquida , ResveratrolRESUMEN
The first phosphorus dendrimers built on a cyclotriphosphazene core and decorated with six or twelve monofluorocyclooctyne units were prepared. A simple stirring allowed the grafting of N-hexyl deoxynojirimycin inhitopes onto their surface by copper-free strain promoted alkyne-azide cycloaddition click reaction. The synthesized iminosugars clusters were tested as multivalent inhibitors of the biologically relevant enzymes ß-glucocerebrosidase and acid α-glucosidase, involved in Gaucher and Pompe lysosomal storage diseases, respectively. For both enzymes, all the multivalent compounds were more potent than the reference N-hexyl deoxynojirimycin. Remarkably, the final dodecavalent compound proved to be one of the best ß-glucocerebrosidase inhibitors described to date. These cyclotriphosphazene-based deoxynojirimycin dendrimers were then evaluated as pharmacological chaperones against Gaucher disease. Not only did these multivalent constructs cross the cell membranes but they were also able to increase ß-glucocerebrosidase activity in Gaucher cells. Notably, dodecavalent compound allowed a 1.4-fold enzyme activity enhancement at a concentration as low as 100â nM. These new monofluorocyclooctyne-presenting dendrimers may further find numerous applications in the synthesis of multivalent objects for biological and pharmacological purposes.
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Dendrímeros , Enfermedad de Gaucher , Humanos , Enfermedad de Gaucher/tratamiento farmacológico , Glucosilceramidasa/metabolismo , Glucosilceramidasa/uso terapéutico , Inhibidores Enzimáticos/metabolismoRESUMEN
Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated.
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Antocianinas , Regulación de la Expresión Génica de las Plantas , Citidina/análogos & derivados , Metilación de ADN/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Perennial woody plants undergo a period of dormancy from the beginning of autumn until the end of spring. Whereas the molecular and physiological events that characterize dormancy release of buds have been described in detail, those occurring in woody tissues underneath the buds are mostly unknown. To bridge this gap, the mRNA populations of cane segments located underneath the bud were analyzed at bud dormancy (E-L 1) and at bud burst (E-L 4). They revealed an important reprogramming of gene expression suggesting that cell division, cell wall metabolism and the mobilization of sugars are the main metabolic and cellular events occurring in cane woody tissues at bud burst. Also, the upregulation of several genes of sugar metabolism, encoding starch- and sucrose-degrading enzymes and sugar transporters, correlates with the decrease in starch and soluble sugars in woody tissues concomitant with increased sucrose synthase and α-amylolytic biochemical activities. The latter is likely due to the VviAMY2 gene that encodes a functional α-amylase as observed after its heterologous expression in yeast. Taken together, these results are consistent with starch and sugar mobilization in canes being primarily involved in grapevine secondary growth initiation and supporting the growth of the emerging bud.
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Pared Celular/metabolismo , Latencia en las Plantas/genética , Latencia en las Plantas/fisiología , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/genética , Vitis/crecimiento & desarrollo , Vitis/genética , Transporte Biológico/genética , Transporte Biológico/fisiología , División Celular/genética , División Celular/fisiología , Pared Celular/genética , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Portugal , ARN Mensajero/metabolismo , Azúcares/metabolismo , alfa-Amilasas/metabolismoRESUMEN
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.
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Linfocitos T CD8-positivos/inmunología , Melanoma/inmunología , Melanoma/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Inmunidad , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/genética , Células TH1/inmunologíaRESUMEN
A concise and asymmetric synthesis of the enantiomeric pyrrolidines 2 and ent-2 are herein reported. Both enantiomers were assessed as ß-GCase inhibitors. While compound ent-2 acted as a poor competitive inhibitor, its enantiomer 2 proved to be a potent non-competitive inhibitor. Docking studies were carried out to substantiate their respective protein binding mode. Both pyrrolidines were also able to enhance lysosomal ß-GCase residual activity in N370S homozygous Gaucher fibroblasts. Notably, the non-competitive inhibitor 2 displayed an enzyme activity enhancement comparable to that of reference compounds IFG and NN-DNJ. This work highlights the impact of inhibitors chirality on their protein binding mode and shows that, beyond competitive inhibitors, the study of non-competitive ones can lead to the identification of new relevant parmacological chaperones.
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Enfermedad de GaucherRESUMEN
Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment. Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment. Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity. Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1. Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer. Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration. Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies. Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.
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Resistencia a Antineoplásicos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Neoplasias Cutáneas/tratamiento farmacológico , Anciano , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Linfocitos T CD8-positivos/patología , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Melanoma/inmunología , Melanoma/mortalidad , Melanoma/patología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones Endogámicos BALB C , Persona de Mediana Edad , Terapia Molecular Dirigida , Nivolumab/uso terapéutico , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Tasa de Supervivencia , Linfocitos T Reguladores/patología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/fisiologíaRESUMEN
Sphingolipid (SL) metabolism alterations have been frequently reported in cancer including in melanoma, a bad-prognosis skin cancer. In normal cells, de novo synthesized ceramide is mainly converted to sphingomyelin (SM), the most abundant SL, by sphingomyelin synthase 1 (SMS1) and, albeit to a lesser extent, SMS2, encoded by the SGMS1 and SGMS2 genes, respectively. Alternatively, ceramide can be converted to glucosylceramide (GlcCer) by the GlcCer synthase (GCS), encoded by the UGCG gene. Herein, we provide evidence for the first time that SMS1 is frequently downregulated in various solid cancers, more particularly in melanoma. Accordingly, various human melanoma cells displayed a SL metabolism signature associated with (i) a robust and a low expression of UGCG and SGMS1/2, respectively, (ii) higher in situ enzyme activity of GCS than SMS, and (iii) higher intracellular levels of GlcCer than SM. SMS1 was expressed at low levels in most of the human melanoma biopsies. In addition, several mutations and increased CpG island methylation in the SGMS1 gene were identified that likely affect SMS1 expression. Finally, low SMS1 expression was associated with a worse prognosis in metastatic melanoma patients. Collectively, our study indicates that SMS1 downregulation in melanoma enhances GlcCer synthesis, triggering an imbalance in the SM/GlcCer homeostasis, which likely contributes to melanoma progression. Evaluating SMS1 expression level in tumor samples might serve as a biomarker to predict clinical outcome in advanced melanoma patients.
RESUMEN
Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.
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Ceramidasa Ácida/metabolismo , Imagen Molecular , Células A549 , Ceramidasa Ácida/antagonistas & inhibidores , Supervivencia Celular , Inhibidores Enzimáticos/farmacología , Humanos , Sondas Moleculares/metabolismoRESUMEN
A series of simple C-alkyl pyrrolidines already known as cytotoxic inhibitors of ceramide glucosylation in melanoma cells can be converted into their corresponding 6-membered analogues by means of a simple ring expansion. This study illustrated how an isomerisation from iminosugar pyrrolidine toward piperidine could invert their targeting from glucosylceramide (GlcCer) formation toward GlcCer hydrolysis. Thus, we found that the 5-membered ring derivatives did not inhibit the hydrolysis reaction of GlcCer catalysed by lysosomal ß-glucocerebrosidase (GBA). On the other hand, the ring-expanded C-alkyl piperidine isomers, non-cytotoxic and inactive regarding ceramide glucosylation, revealed to be potent inhibitors of GBA. A molecular docking study showed that the positions of the piperidine ring of the compound 6b and its analogous 2-O-heptyl DIX 8 were similar to that of isofagomine. Furthermore, compound 6b promoted mutant GBA enhancements over 3-fold equivalent to that of the related O-Hept DIX 8 belonging to one of the most potent iminosugar-based pharmacological chaperone series reported to date.
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Ceramidas/química , Inhibidores Enzimáticos/química , Glucosilceramidasa/antagonistas & inhibidores , Iminoazúcares/química , Animales , Células Cultivadas , Fibroblastos/efectos de los fármacos , Glucosilceramidasa/metabolismo , Humanos , Hidrólisis , Iminopiranosas/química , Isomerismo , Lisosomas , Melanoma Experimental , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Piperidinas/química , Unión Proteica , Pirrolidinas/química , Relación Estructura-ActividadRESUMEN
Phenotypic plasticity and subsequent generation of intratumoral heterogeneity underly key traits in malignant melanoma such as drug resistance and metastasis. Melanoma plasticity promotes a switch between proliferative and invasive phenotypes characterized by different transcriptional programs of which MITF is a critical regulator. Here, we show that the acid ceramidase ASAH1, which controls sphingolipid metabolism, acted as a rheostat of the phenotypic switch in melanoma cells. Low ASAH1 expression was associated with an invasive behavior mediated by activation of the integrin alphavbeta5-FAK signaling cascade. In line with that, human melanoma biopsies revealed heterogeneous staining of ASAH1 and low ASAH1 expression at the melanoma invasive front. We also identified ASAH1 as a new target of MITF, thereby involving MITF in the regulation of sphingolipid metabolism. Together, our findings provide new cues to the mechanisms underlying the phenotypic plasticity of melanoma cells and identify new anti-metastatic targets.
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Ceramidasa Ácida/genética , Proliferación Celular/genética , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Línea Celular Tumoral , Femenino , Quinasa 1 de Adhesión Focal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Melanoma/patología , Invasividad Neoplásica/genética , Proteínas Proto-Oncogénicas B-raf , Receptores de Vitronectina/genética , Transducción de SeñalRESUMEN
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.
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Compuestos de Bifenilo/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Melanoma/tratamiento farmacológico , Nitrofenoles/administración & dosificación , Receptores de Lisoesfingolípidos/metabolismo , Esfingolípidos/sangre , Sulfonamidas/administración & dosificación , Animales , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Lisofosfolípidos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Ratones , Nitrofenoles/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piperazinas/administración & dosificación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Sulfonamidas/farmacología , Vemurafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes-ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream.
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The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.
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Análisis Mutacional de ADN/métodos , Metanosulfonato de Etilo/metabolismo , Mutación , Solanum lycopersicum/genética , Variación Genética , Factores de TiempoRESUMEN
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-ß1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-ß1 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.
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Macrófagos/fisiología , Melanoma/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular , Proliferación Celular , Regulación hacia Abajo , Humanos , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Receptores de Lisoesfingolípidos/fisiología , Receptores de Esfingosina-1-Fosfato , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
OBJECTIVE: To establish a diagnosis and provide counseling and treatment for 3 adult patients from one family presenting with peripheral osteolysis. METHODS: Following clinical and radiographic assessment, exome sequencing, targeted gene resequencing, and determination of enzyme activity in cultured fibroblasts were performed. RESULTS: The proband (age 40 years) had a history of episodic fever and pain in childhood that subsided around puberty. He and 2 of his older sisters (ages 58 and 60 years, respectively) showed adult-onset progressive shortening of fingers and toes with redundancy of the overlying skin. Radiographs showed severe osteolysis of the distal radius and ulna, carpal bones, metacarpal bones, and phalanges. Sequencing of the known genes for recessively inherited osteolysis, MMP2 and MMP14, failed to show pathogenic mutations. Exome sequencing revealed compound heterozygosity for mutations c.505T>C (p.Trp169Arg) and c.760A>G (p.Arg254Gly) in ASAH1, the gene coding for acid ceramidase. Sanger sequencing confirmed correct segregation in the family, and enzyme activity in fibroblast cultures from the patients was reduced to â¼8% of that in controls, confirming a diagnosis of Farber's disease. CONCLUSION: Our findings indicate that hypomorphic mutations in ASAH1 may result in an osteoarticular phenotype with a juvenile phase resembling rheumatoid arthritis that evolves to osteolysis as the final stage in the absence of neurologic signs. This observation delineates a novel type of recessively inherited peripheral osteolysis and illustrates the long-term skeletal manifestations of acid ceramidase deficiency (Farber's disease) in what appear to be the oldest affected individuals known so far.