RESUMO
Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.
Assuntos
Citocinas , Redes Reguladoras de Genes , Citocinas/genética , Proteômica , Fator de Transcrição GATA1/metabolismo , Diferenciação Celular/genética , Ceramidas , HomeostaseRESUMO
Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Esfingosina/metabolismo , Neoplasias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.
Assuntos
Ceramidas , Ceramidase Neutra , Domínio Catalítico , Ceramidas/química , Ceramidase Neutra/antagonistas & inibidores , Esfingosina/químicaRESUMO
BACKGROUND: Colorectal cancer (CRC) is increasing in low- and middle-income countries, likely due to changing lifestyle habits, including diet. We aimed to investigate the relationship between dietary betaine, choline, and choline-containing compounds and CRC risk. METHODS: We analyzed data from a case-control study, including 865 CRC cases and 3206 controls from Iran. Detailed information was collected by trained interviewers using validated questionnaires. The intake of free choline, phosphocholine (Pcho), glycerophosphocholine (GPC), phosphatidylcholine (PtdCho), and sphingomyelin (SM), as well as of betaine was estimated from food frequency questionnaires and categorized into quartiles. The odds ratios (OR) and 95% confidence intervals (CI) of CRC for choline and betaine quartiles were calculated using multivariate logistic regression by adjusting for potential confounders. RESULTS: We observed excess risk of CRC in the highest versus lowest intake of total choline (OR = 1.23, 95% CI 1.13, 1.33), GPC (OR = 1.13, 95% CI 1.00, 1.27), and SM (OR = 1.14, 95% CI 1.01, 1.28). The intake of betaine exerted an inverse association with CRC risk (OR = 0.91, 95% CI 0.83, 0.99). There was no association between free choline, Pcho, PtdCho, and CRC. Analyses stratified by gender showed an elevated OR of CRC in men for SM intake OR = 1.20, 95% CI 1.03, 1.40) and a significantly decreased CRC risk in women for betaine intake (OR = 0.84, 95% CI 0.73, 0.97). CONCLUSION: Dietary modifications leading to an increase in betaine sources and managing the use of animal products as references for SM or other choline types might contribute to decreasing the risk of CRC.
RESUMO
Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Regulação para Cima , Proteína Supressora de Tumor p53/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingolipídeos , Doxorrubicina/farmacologia , Esfingosina/farmacologia , Esfingosina/metabolismo , Lisofosfolipídeos/farmacologiaRESUMO
Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.
Assuntos
Injúria Renal Aguda , Lipogranulomatose de Farber , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Animais , Apoptose/fisiologia , Cisplatino/efeitos adversos , Fibroblastos/metabolismo , Humanos , Camundongos , Ceramidase Neutra/metabolismoRESUMO
Cancer cells may depend on exogenous serine, depletion of which results in slower growth and activation of adaptive metabolic changes. We previously demonstrated that serine and glycine (SG) deprivation causes loss of sphingosine kinase 1 (SK1) in cancer cells, thereby increasing the levels of its lipid substrate, sphingosine (Sph), which mediates several adaptive biological responses. However, the signaling molecules regulating SK1 and Sph levels in response to SG deprivation have yet to be defined. Here, we identify 1-deoxysphinganine (dSA), a noncanonical sphingoid base generated in the absence of serine from the alternative condensation of alanine and palmitoyl CoA by serine palmitoyl transferase, as a proximal mediator of SG deprivation in SK1 loss and Sph level elevation upon SG deprivation in cancer cells. SG starvation increased dSA levels in vitro and in vivo and in turn induced SK1 degradation through a serine palmitoyl transferase-dependent mechanism, thereby increasing Sph levels. Addition of exogenous dSA caused a moderate increase in intracellular reactive oxygen species, which in turn decreased pyruvate kinase PKM2 activity while increasing phosphoglycerate dehydrogenase levels, and thereby promoted serine synthesis. We further showed that increased dSA induces the adaptive cellular and metabolic functions in the response of cells to decreased availability of serine likely by increasing Sph levels. Thus, we conclude that dSA functions as an initial sensor of serine loss, SK1 functions as its direct target, and Sph functions as a downstream effector of cellular and metabolic adaptations. These studies define a previously unrecognized "physiological" nontoxic function for dSA.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)RESUMO
A high-fat diet (HFD) and obesity are risk factors for many diseases including breast cancer. This is particularly important with close to 40% of the current adult population being overweight or obese. Previous studies have implicated that Mediterranean diets (MDs) partially protect against breast cancer. However, to date, the links between diet and breast cancer progression are not well defined. Therefore, to begin to define and assess this, we used an isocaloric control diet (CD) and two HFDs enriched with either olive oil (OOBD, high in oleate, and unsaturated fatty acid in MDs) or a milk fat-based diet (MFBD, high in palmitate and myristate, saturated fatty acids in Western diets) in a mammary polyomavirus middle T antigen mouse model (MMTV-PyMT) of breast cancer. Our data demonstrate that neither MFBD or OOBD altered the growth of primary tumors in the MMTV-PyMT mice. The examination of lung metastases revealed that OOBD mice exhibited fewer surface nodules and smaller metastases when compared to MFBD and CD mice. These data suggest that different fatty acids found in different sources of HFDs may alter breast cancer metastasis.
Assuntos
Neoplasias da Mama/patologia , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/toxicidade , Ácidos Graxos/toxicidade , Neoplasias Pulmonares/secundário , Leite/toxicidade , Ração Animal , Animais , Antígenos Transformantes de Poliomavirus , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Azeite de Oliva/toxicidade , Medição de Risco , Fatores de Risco , Carga Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Doxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed. METHODS: Effects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox. RESULTS: The results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK-including Fyn, Yes, and Src-partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells. CONCLUSIONS: Overall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.
Assuntos
Movimento Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Quinases da Família src/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Relação Dose-Resposta a Droga , Feminino , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase C/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.
Assuntos
Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Infecções por Retroviridae/metabolismo , Infecções Tumorais por Vírus/metabolismo , Animais , Carcinogênese , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos KnockoutRESUMO
Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1ß and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.
Assuntos
Inflamação/imunologia , Monócitos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Fator de Necrose Tumoral alfa/efeitos adversos , Ceramidas/metabolismo , Humanos , Inflamação/terapia , Terapia de Alvo Molecular , Monócitos/enzimologia , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células THP-1RESUMO
Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.
Assuntos
Proliferação de Células , Glicoesfingolipídeos/biossíntese , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Células Cultivadas , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Melanoma/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade , Imunoterapia , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Células Th1/imunologiaRESUMO
We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models.
Assuntos
Ceramidas/fisiologia , Doxorrubicina/farmacologia , Proteína Fosfatase 1/fisiologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células HeLa , HumanosRESUMO
Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.
Assuntos
Neoplasias do Colo/imunologia , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Ceramidase Neutra/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral/transplante , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Ferroptose/efeitos dos fármacos , Ferroptose/imunologia , Humanos , Concentração Inibidora 50 , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Simulação de Acoplamento Molecular , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Ceramidase Neutra/antagonistas & inibidores , Ceramidase Neutra/genética , Estabilidade Proteica/efeitos dos fármacos , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Receptores de Esfingosina-1-Fosfato , Diferenciação Celular , Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMO
Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C24:1 -ceramide for the formation of lamellipodia. These results suggested that C24:1 -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C24:1 -ceramide. Collectively, the CerS2-C24:1 -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.
Assuntos
Movimento Celular , Ceramidas/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/metabolismo , Pseudópodes/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Ceramidas/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Neoplasias Ovarianas/patologia , Pseudópodes/efeitos dos fármacos , Esfingosina N-Aciltransferase/antagonistas & inibidores , Esfingosina N-Aciltransferase/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.
Assuntos
Ceramidas/metabolismo , Ferroptose/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Autofagia , Humanos , Camundongos , TransfecçãoRESUMO
Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence-as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated ß-galactosidase activity (SA-ß-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-ß-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.