RESUMEN

Prostate cancer (PCa) development and progression relies on the programming of glucose and lipid metabolism, and this involves alterations in androgen receptor expression and signalling. Defining the molecular mechanism that underpins this metabolic programming will have direct significance for patients with PCa who have a poor prognosis. Here we show that there is a dynamic balance between sortilin and syndecan-1, that reports on different metabolic phenotypes. Using tissue microarrays, we demonstrated by immunohistochemistry that sortilin was highly expressed in low-grade cancer, while syndecan-1 was upregulated in high-grade disease. Mechanistic studies in prostate cell lines revealed that in androgen-sensitive LNCaP cells, sortilin enhanced glucose metabolism by regulating GLUT1 and GLUT4, while binding progranulin and lipoprotein lipase (LPL) to limit lipid metabolism. In contrast, in androgen-insensitive PC3 cells, syndecan-1 was upregulated, interacted with LPL and colocalised with ß3 integrin to promote lipid metabolism. In addition, androgen-deprived LNCaP cells had decreased expression of sortilin and reduced glucose-metabolism, but increased syndecan-1 expression, facilitating interactions with LPL and possibly ß3 integrin. We report a hitherto unappreciated molecular mechanism for PCa, which may have significance for disease progression and how androgen-deprivation therapy might promote castration-resistant PCa.

Asunto(s)

Neoplasias de la Próstata , Masculino , Humanos , Próstata , Sindecano-1/genética , Antagonistas de Andrógenos , Andrógenos , Integrina beta3 , Procesos Neoplásicos

RESUMEN

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Asunto(s)

Antineoplásicos , Leucemia Mieloide Aguda , Antineoplásicos/uso terapéutico , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Ceramidas/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo

RESUMEN

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.

Asunto(s)

Calreticulina , Trastornos Mieloproliferativos , Animales , Anticuerpos Monoclonales , Calreticulina/genética , Calreticulina/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Ratas

RESUMEN

Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors. Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P2 and S1P4) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P2/4, inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P2/4.

Asunto(s)

Pirazoles/química , Piridinas/química , Esfingolípidos/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Receptores de Esfingosina-1-Fosfato/metabolismo , Células HEK293 , Humanos , Cinética , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Receptores de Esfingosina-1-Fosfato/genética

RESUMEN

Pre-treatments are usually necessary to prepare biowaste to anaerobic digestion. The major objectives are (i) to remove undesirable materials such as plastics and metals, which may contaminate the biowaste even if separated source-collection systems are implemented, and (ii) to extract the most readily biodegradable organic fractions from the waste stream. In this study, two wet mechanical pre-treatments, namely air-compressed press and worm screw press, were investigated on urban household biowaste. Two liquid to solid ratios were tested in each pre-treatment. Anaerobic digestion of pre-treated biowaste was studied by measuring their biomethane potentials and by controlled experiments in a continuously stirred-tank reactor with a feed load of 3.5 gVS.L-1.d-1. It was observed that increasing liquid to solid ratio in the pre-treatments allowed to increase the proportion of biodegradable organic matter extracted from the biowaste, up to 949 gCOD.kgTS-1 from household biowaste. The biomethane potentials of pre-treated waste were very high (up 525 LCH4.kgVS-1) and COD (949 gCOD.kg-1TS) from household biowaste. Anaerobic digestion in continuously stirred-tank reactor showed a very strong conversion of COD load (81%) and a high methane production up to 345 LCH4.kgVS-1.

Asunto(s)

Reactores Biológicos , Eliminación de Residuos , Anaerobiosis , Metano , Fenómenos Físicos

RESUMEN

The present study investigated a wet mechanical pretreatment to improve methane production by anaerobic digestion from biowaste material by separating a biodegradable aqueous slurry fraction (ASF) from a more recalcitrant particulate fraction (PF). Four source-sorted municipal biowastes were studied, namely household (HBW), supermarket (SBW), restaurant (RBW), and green biowaste (GBW). The treatment consisted in soaking the waste in water and then pressing the slurry through a grid with 3-mm openings to separate the two fractions. Methane production of ASF and PF obtained from the four biowastes were measured using the BMP protocol and compared to the potential of the respective untreated biowaste. Results were very different for GBW as compared to the other three BWs. With GBW, which was the most lignocellulosic of the BW studied, only 17% of the initial methane potential was recovered in the ASF. The extraction was much better on the other biowastes and increased in the following order: HBW (58%) ≃ RBW (57%) < SBW (67%). The ASF from these biowastes exhibited low total solid contents and high BMPs (416, 408, and 423 NLCH4.g-1vs for HBW, RBW, and SBW respectively). The experimental results obtained in this study therefore showed that wet pressing separation was an efficient pretreatment to improve and facilitate methane production by anaerobic digestion of biowaste such as HBW, RBW, and SBW.

Asunto(s)

Eliminación de Residuos , Anaerobiosis , Reactores Biológicos , Metano , Agua

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RESUMEN

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.

Asunto(s)

Neoplasias de la Mama/patología , Fibroblastos Asociados al Cáncer/patología , Moléculas de Adhesión Celular/metabolismo , Reprogramación Celular , Proteínas de la Matriz Extracelular/metabolismo , eIF-2 Quinasa/metabolismo , Quinasas Asociadas a rho/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Moléculas de Adhesión Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Ratones , Comunicación Paracrina , eIF-2 Quinasa/genética , Quinasas Asociadas a rho/genética

RESUMEN

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

Asunto(s)

Proteínas Portadoras/metabolismo , Lisina/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Secuencias de Aminoácidos , Proteínas Portadoras/genética , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Humanos , Lisina/genética , Proteínas de Microfilamentos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteolisis , Ubiquitinación

RESUMEN

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.

Asunto(s)

Dineínas Citoplasmáticas/genética , Genes Supresores de Tumor , Glioblastoma/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Apoptosis/genética , Carcinogénesis/genética , Línea Celular Tumoral , Membrana Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Células HEK293 , Humanos , Lisofosfolípidos/genética , Ratones , Esfingosina/análogos & derivados , Esfingosina/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The integrated fixed-film activated sludge (IFAS) process is being increasingly used to enhance nitrogen removal for former activated sludge systems. The aim of this work is to evaluate a numerical model of a new nitrifying/denitrifying IFAS configuration. It consists of two carrier-free reactors (anoxic and aerobic) and one IFAS reactor with a filling ratio of 43% of carriers, followed by a clarifier. Simulations were carried out with GPS-X involving the nitrification reaction combined with a 1D heterogeneous biofilm model, including attachment/detachment processes. An original iterative calibration protocol was created comprising four steps and nine actions. Experimental campaigns were carried out to collect data on the pilot in operation, specifically for modelling purpose. The model used was able to predict properly the variations of the activated sludge (bulk) and the biofilm masses, the nitrification rates of both the activated sludge and the biofilm, and the nitrogen concentration in the effluent for short (4-10 days) and long (300 days) simulation runs. A calibrated parameter set is proposed (biokinetics, detachment, diffusion) related to the activated sludge, the biofilm and the effluent variables to enhance the model prediction on hourly and daily data sets.

Asunto(s)

Biopelículas/crecimiento & desarrollo , Modelos Biológicos , Nitrógeno/metabolismo , Aguas del Alcantarillado/microbiología , Purificación del Agua/métodos

RESUMEN

Sphingosine kinase 1 (SK1) is a key regulator of the cellular balance between proapoptotic and prosurvival sphingolipids. Oncogenic signaling by SK1 relies on its localization to the plasma membrane, which is mediated by the calcium and integrin binding protein CIB1 via its Ca2+-myristoyl switch function. Here we show that another member of the CIB family, CIB2, plays a surprisingly opposite role to CIB1 in the regulation of SK1 signaling. CIB2 bound SK1 on the same site as CIB1, yet it lacks the Ca2+-myristoyl switch function. As a result, CIB2 blocked translocation of SK1 to the plasma membrane and inhibited its subsequent signaling, which included sensitization to TNFα-induced apoptosis and inhibition of Ras-induced neoplastic transformation. CIB2 was significantly downregulated in ovarian cancer and low CIB2 expression was associated with poor prognosis in ovarian cancer patients. Notably, reintroduction of CIB2 in ovarian cancer cells blocked plasma membrane localization of endogenous SK1, reduced in vitro neoplastic growth and tumor growth in mice, and suppressed cell motility and invasiveness both in vitro and in vivo Consistent with the in vitro synergistic effects between the SK1-specific inhibitor SK1-I and standard chemotherapeutics, expression of CIB2 also sensitized ovarian cancer cells to carboplatin. Together, these findings identify CIB2 as a novel endogenous suppressor of SK1 signaling and potential prognostic marker and demonstrate the therapeutic potential of SK1 in this gynecologic malignancy. Cancer Res; 77(18); 4823-34. ©2017 AACR.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Proteínas de Unión al Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proteínas de Unión al Calcio/genética , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro. The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.

Asunto(s)

Antineoplásicos/farmacología , Bortezomib/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Mieloma Múltiple/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Modelos Biológicos , Terapia Molecular Dirigida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Inhibidores de Proteasoma/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML.

Asunto(s)

Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Clorometilcetonas de Aminoácidos/farmacología , Amino Alcoholes/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Inhibidores de Caspasas/farmacología , Caspasas/genética , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos NOD , Terapia Molecular Dirigida , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an anti-proliferative/pro-apoptotic function for SK2, while others indicate it has a pro-survival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.

Asunto(s)

Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Apoptosis , Carcinogénesis , Proliferación Celular , Supervivencia Celular , Ceramidas/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Transporte de Proteínas , Esfingosina/análogos & derivados , Esfingosina/metabolismo

RESUMEN

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

Asunto(s)

Proteínas 14-3-3/metabolismo , Proliferación Celular/fisiología , Homeostasis/fisiología , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , Quinasas Asociadas a rho/metabolismo , Animales , Epidermis/metabolismo , Ratones

RESUMEN

Sphingosine 1-phosphate (S1P) is a bioactive lipid that can function both extracellularly and intracellularly to mediate a variety of cellular processes. Using lipid affinity matrices and a radiolabeled lipid binding assay, we reveal that S1P directly interacts with the transcription factor peroxisome proliferator-activated receptor (PPAR)γ. Herein, we show that S1P treatment of human endothelial cells (ECs) activated a luciferase-tagged PPARγ-specific gene reporter by ∼12-fold, independent of the S1P receptors. More specifically, in silico docking, gene reporter, and binding assays revealed that His323 of the PPARγ ligand binding domain is important for binding to S1P. PPARγ functions when associated with coregulatory proteins, and herein we identify that peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1)ß binds to PPARγ in ECs and their progenitors (nonadherent endothelial forming cells) and that the formation of this PPARγ:PGC1ß complex is increased in response to S1P. ECs treated with S1P selectively regulated known PPARγ target genes with PGC1ß and plasminogen-activated inhibitor-1 being increased, no change to adipocyte fatty acid binding protein 2 and suppression of CD36. S1P-induced in vitro tube formation was significantly attenuated in the presence of the PPARγ antagonist GW9662, and in vivo application of GW9662 also reduced vascular development in Matrigel plugs. Interestingly, activation of PPARγ by the synthetic ligand troglitazone also reduced tube formation in vitro and in vivo. To support this, Sphk1(-/-)Sphk2(+/-) mice, with low circulating S1P levels, demonstrated a similar reduction in vascular development. Taken together, our data reveal that the transcription factor, PPARγ, is a bona fide intracellular target for S1P and thus suggest that the S1P:PPARγ:PGC1ß complex may be a useful target to manipulate neovascularization.

Asunto(s)

Células Endoteliales de la Vena Umbilical Humana/metabolismo , Lisofosfolípidos/metabolismo , Neovascularización Fisiológica/fisiología , PPAR gamma/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Lisofosfolípidos/genética , Ratones , Ratones Noqueados , PPAR gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteínas de Unión al ARN , Receptores de Lisoesfingolípidos/genética , Serpina E2/genética , Serpina E2/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células U937

RESUMEN

The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Adenosina Trifosfato/química , Antineoplásicos/química , Inhibidores Enzimáticos/química , Neoplasias Pulmonares/tratamiento farmacológico , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Adenocarcinoma/metabolismo , Animales , Apoptosis , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Transgénicos , Conformación Molecular , Mutagénesis , Mutación , Trasplante de Neoplasias , Neovascularización Patológica , Unión Proteica , Esfingolípidos/química

RESUMEN

OBJECTIVES: The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. METHODS: The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. RESULTS: Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. CONCLUSIONS: These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.

Asunto(s)

Desdiferenciación Celular , Células Endoteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Células Madre/enzimología , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Células Endoteliales/citología , Células HEK293 , Humanos , Lentivirus , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Células Madre/citología , Transducción Genética

RESUMEN

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.

Asunto(s)

Membrana Celular/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Transformación Celular Neoplásica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Células 3T3 NIH , Fosforilación , Unión Proteica , Transporte de Proteínas