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ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.
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Anticuerpos Monoclonales Humanizados , Inmunoterapia , Índice Terapéutico , Antígenos CD19 , Inmunoterapia Adoptiva/métodosRESUMEN
Idiopathic intracranial hypertension (IIH) is characterized by raised intracranial pressure with unknown etiology. The most common neurological manifestations are headache and visual loss. Often, other cranial nerve impairments are also found, most commonly in the VI nerve. Trigeminal neuralgia (TN) is a debilitating condition that is most frequently caused by neurovascular pathology, but TN secondary to IIH is a rare and poorly described topic. Possible explanations of TN in these patients include the distortion of the local anatomy at CN entry zones and fluid displacement causing distortion of the Meckel's cave. In the case below we describe the clinical course of an obese female patient with TN-like pain who underwent a ventriculoperitoneal shunt to treat IIH and experienced complete resolution of both conditions.
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The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Tirosina Quinasa del Receptor Axl , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Linfocitos T , Receptores de Antígenos de Linfocitos T/genética , Xenoinjertos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inmunoterapia Adoptiva/métodosRESUMEN
Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos , Neoplasias , Citocinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Activación de Linfocitos , Linfocitos TRESUMEN
Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.
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Fibroblastos Asociados al Cáncer , Mieloma Múltiple , Médula Ósea , Fibroblastos Asociados al Cáncer/patología , Tratamiento Basado en Trasplante de Células y Tejidos , Fibroblastos , Humanos , Inmunoterapia Adoptiva/métodos , Mieloma Múltiple/patología , Microambiente TumoralRESUMEN
Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.
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Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Simportadores/análisis , Animales , Antígenos CD19 , Modelos Animales de Enfermedad , Femenino , Humanos , Células K562 , Masculino , Neoplasias/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.
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Antígeno B7-H1/sangre , Leucemia Linfocítica Crónica de Células B/terapia , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Antígeno B7-H1/genética , Línea Celular Tumoral , Vesículas Extracelulares/genética , Vesículas Extracelulares/inmunología , Femenino , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Receptores de Antígenos de Linfocitos T/sangre , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these "side" effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.
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Antineoplásicos/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ceramidas/farmacología , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células HeLa , Humanos , Fosforilación/efectos de los fármacos , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann-Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics.
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Inhibidores Enzimáticos/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielinas/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Animales , HumanosRESUMEN
Ceramide synthases (CerS1-CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death.
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Apoptosis , Ceramidas/biosíntesis , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Oxidorreductasas/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Caspasas/metabolismo , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Fumonisinas/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Células MCF-7 , Oxidorreductasas/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies.
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Proteínas del Citoesqueleto/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Comunicación Autocrina/genética , Proteínas del Citoesqueleto/genética , Factor de Crecimiento Epidérmico/genética , Células HeLa , Humanos , Lisofosfolípidos/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Fosforilación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was â¼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an â¼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an â¼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.
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Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/etiología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Regulación hacia Abajo , Quinasa 1 de Adhesión Focal/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Renales/etiología , Neoplasias Renales/patología , Lisofosfolípidos/biosíntesis , Invasividad Neoplásica , Neovascularización Patológica , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/biosíntesis , Receptores de Esfingosina-1-Fosfato , Regulación hacia ArribaRESUMEN
Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid ß-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4ß-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid ß-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.
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Interleucina-6/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Desipramina/farmacología , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Interleucina-6/genética , Células MCF-7 , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Enfermedades de Niemann-Pick/genética , Enfermedades de Niemann-Pick/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
A key but poorly studied domain of sphingolipid functions encompasses endocytosis, exocytosis, cellular trafficking, and cell movement. Recently, the ezrin, radixin and moesin (ERM) family of proteins emerged as novel potent targets regulated by sphingolipids. ERMs are structural proteins linking the actin cytoskeleton to the plasma membrane, also forming a scaffold for signaling pathways that are used for cell proliferation, migration and invasion, and cell division. Opposing functions of the bioactive sphingolipid ceramide and sphingosine-1-phosphate (S1P), contribute to ERM regulation. S1P robustly activates whereas ceramide potently deactivates ERM via phosphorylation/dephosphorylation, respectively. This recent dimension of cytoskeletal regulation by sphingolipids opens up new avenues to target cell dynamics, and provides further understanding of some of the unexplained biological effects mediated by sphingolipids. In addition, these studies are providing novel inroads into defining basic mechanisms of regulation and action of bioactive sphingolipids. This review describes the current understanding of sphingolipid regulation of the cytoskeleton, it also describes the biologies in which ERM proteins have been involved, and finally how these two large fields have started to converge. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
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Fenómenos Fisiológicos Celulares , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Esfingolípidos/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCßII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCßII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCßII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCßII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.
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Complejos Multiproteicos/metabolismo , Fosfolipasa D/metabolismo , Proteína Quinasa C beta/metabolismo , Proteína Quinasa C/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular , Endocitosis/genética , Endocitosis/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Fosfolipasa D/genética , Proteína Quinasa C/genética , Proteína Quinasa C beta/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Sphingosine kinase 1 (SK1) produces the pro-survival sphingolipid sphingosine 1-phosphate and has been implicated in inflammation, proliferation, and angiogenesis. Recent studies identified TRAF2 as a sphingosine 1-phosphate target, implicating SK1 in activation of the NF-κB pathway, but the functional consequences of this connection on gene expression are unknown. Here, we find that loss of SK1 potentiates induction of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted; also known as CCL5) in HeLa cells stimulated with TNF-α despite RANTES induction being highly dependent on the NF-κB pathway. Additionally, we find that SK1 is not required for TNF-induced IKK phosphorylation, IκB degradation, nuclear translocation of NF-κB subunits, and transcriptional NF-κB activity. In contrast, loss of SK1 prevented TNF-induced phosphorylation of p38 MAPK, and inhibition of p38 MAPK, like SK1 knockdown, also potentiates RANTES induction. Finally, in addition to RANTES, loss of SK1 also potentiated the induction of multiple chemokines and cytokines in the TNF response. Taken together, these data identify a potential and novel anti-inflammatory function of SK1 in which chemokine levels are suppressed through SK1-mediated activation of p38 MAPK. Furthermore, in this system, activation of NF-κB is dissociated from SK1, suggesting that the interaction between these pathways may be more complex than currently thought.
Asunto(s)
Quimiocina CCL5/biosíntesis , FN-kappa B/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Quimiocina CCL5/genética , Activación Enzimática/fisiología , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/genética , Fosforilación/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in cell proliferation, angiogenesis, inflammation and malignant transformation among other functions. S1P acts either directly on intracellular targets or activates G protein-coupled receptors, specifically five S1P receptors (S1PRs). The identified S1PRs differ in cellular and tissue distribution, and each is coupled to specific G proteins, which mediate unique functions. Here, we describe functional characteristics of all five receptors, emphasizing S1PR2, which is critical in the immune, nervous, metabolic, cardiovascular, musculoskeletal, and renal systems. This review also describes the role of this receptor in tumor growth and metastasis and suggests potential therapeutic avenues that exploit S1PR2.
Asunto(s)
Lisofosfolípidos/metabolismo , Neoplasias/patología , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Humanos , Neoplasias/metabolismo , Esfingosina/metabolismoRESUMEN
Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.