RESUMEN
Purinergic signaling plays an important role in regulating bladder contractility and voiding. Abnormal purinergic signaling is associated with lower urinary tract symptoms (LUTS). Ecto-5'-nucleotidase (NT5E) catalyzes dephosphorylation of extracellular AMP to adenosine, which in turn promotes adenosine-A2b receptor signaling to relax bladder smooth muscle (BSM). The functional importance of this mechanism was investigated using Nt5e knockout (Nt5eKO) mice. Increased voiding frequency of small voids revealed by voiding spot assay was corroborated by urodynamic studies showing shortened voiding intervals and decreased bladder compliance. Myography indicated reduced contractility of Nt5eKO BSM. These data support a role for NT5E in regulating bladder function through modulation of BSM contraction and relaxation. However, the abnormal bladder phenotype of Nt5eKO mice is much milder than we previously reported in A2b receptor knockout (A2bKO) mice, suggesting compensatory response(s) in Nt5eKO mouse bladder. To better understand this compensatory mechanism, we analyzed changes in purinergic and other receptors controlling BSM contraction and relaxation in the Nt5eKO bladder. We found that the relative abundance of muscarinic CHRM3 (cholinergic receptor muscarinic 3), purinergic P2X1, and A2b receptors was unchanged, whereas P2Y12 receptor was significantly downregulated, suggesting a negative feedback response to elevated ADP signaling. Further studies of additional ecto-nucleotidases indicated significant upregulation of the nonspecific urothelial alkaline phosphatase ALPL, which might mitigate the degree of voiding dysfunction by compensating for Nt5e deletion. These data suggest a mechanistic complexity of the purinergic signaling network in bladder and imply a paracrine mechanism in which urothelium-released ATP and its rapidly produced metabolites coordinately regulate BSM contraction and relaxation.
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5'-Nucleotidasa , Vejiga Urinaria , Animales , Ratones , 5'-Nucleotidasa/genética , Adenosina , Fosfatasa Alcalina , Colinérgicos , Ratones NoqueadosRESUMEN
The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.
Asunto(s)
Esquizofrenia , Humanos , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Estudio de Asociación del Genoma Completo , Células HEK293 , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Neuronas/fisiología , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMEN
BACKGROUND: Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle-brachial index (ABI). METHODS: Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams. RESULTS: Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort. CONCLUSIONS: Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort.(ClinicalTrials.gov Identifier NCT01585402).
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5'-Nucleotidasa , Ácido Etidrónico , Proteínas Ligadas a GPI , Calcificación Vascular , Humanos , Proyectos Piloto , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/diagnóstico por imagen , Ácido Etidrónico/uso terapéutico , Ácido Etidrónico/efectos adversos , Masculino , Femenino , Persona de Mediana Edad , Resultado del Tratamiento , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/deficiencia , Factores de Tiempo , Proteínas Ligadas a GPI/sangre , Índice Tobillo Braquial , Adulto , Conservadores de la Densidad Ósea/uso terapéutico , Conservadores de la Densidad Ósea/efectos adversos , Progresión de la Enfermedad , Enfermedad Arterial Periférica/tratamiento farmacológico , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/fisiopatología , Anciano , Extremidad Inferior/irrigación sanguínea , Angiografía por Tomografía Computarizada , Predisposición Genética a la Enfermedad , Flujo Sanguíneo RegionalRESUMEN
The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5'nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.
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Astrocitos , Corteza Cerebral , Hipocampo , Ratones Endogámicos BALB C , Derrota Social , Estrés Psicológico , Animales , Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Estrés Psicológico/metabolismo , Masculino , Ratones , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Conexina 43/metabolismo , Conexina 43/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Viral infections trigger inflammation by controlling ATP release. CD39 ectoenzymes hydrolyze ATP/ADP to AMP, which is converted by CD73 into anti-inflammatory adenosine (ADO). ADO is an anti-inflammatory and immunosuppressant molecule which can enhance viral persistence and severity. The CD39-CD73-adenosine axis contributes to the immunosuppressive T-reg microenvironment and may affect COVID-19 disease progression. Here, we investigated the link between CD39 expression, mostly on T-regs, and levels of CD73, adenosine, and adenosine receptors with COVID-19 severity and progression. Our study included 73 hospitalized COVID-19 patients, of which 33 were moderately affected and 40 suffered from severe infection. A flow cytometric analysis was used to analyze the frequency of T-regulatory cells (T-regs), CD39+ T-regs, and CD39+CD4+ T-cells. Plasma concentrations of adenosine, IL-10, and TGF-ß were quantified via an ELISA. An RT-qPCR was used to analyze the gene expression of CD73 and adenosine receptors (A1, A2A, A2B, and A3). T-reg cells were higher in COVID-19 patients compared to healthy controls (7.4 ± 0.79 vs. 2.4 ± 0.28; p < 0.0001). Patients also had a higher frequency of the CD39+ T-reg subset. In addition, patients who suffered from a severe form of the disease had higher CD39+ T-regs compared with moderately infected patients. CD39+CD4+ T cells were increased in patients compared to the control group. An analysis of serum adenosine levels showed a marked decrease in their levels in patients, particularly those suffering from severe illness. However, this was paralleled with a marked decline in the expression levels of CD73. IL-10 and TGF-ß levels were higher in COVID-19; in addition, their values were also higher in the severe group. In conclusion, there are distinct immunological alterations in CD39+ lymphocyte subsets and a dysregulation in the adenosine signaling pathway in COVID-19 patients which may contribute to immune dysfunction and disease progression. Understanding these immunological alterations in the different immune cell subsets and adenosine signaling provides valuable insights into the pathogenesis of the disease and may contribute to the development of novel therapeutic approaches targeting specific immune mechanisms.
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Adenosina , COVID-19 , Linfocitos T Reguladores , Humanos , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Antiinflamatorios , Antígenos CD/genética , Antígenos CD/metabolismo , Progresión de la Enfermedad , Interleucina-10 , Receptores Purinérgicos P1/genética , Factor de Crecimiento Transformador beta/genética , Linfocitos T Reguladores/metabolismoAsunto(s)
5'-Nucleotidasa , Mano , Valor Predictivo de las Pruebas , Calcificación Vascular , Humanos , Calcificación Vascular/diagnóstico por imagen , 5'-Nucleotidasa/deficiencia , 5'-Nucleotidasa/genética , Mano/irrigación sanguínea , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/metabolismo , Angiografía por Tomografía Computarizada , Masculino , Pierna/irrigación sanguínea , FemeninoRESUMEN
Metastasis and limited benefits of immune checkpoint blockade are two obstacles to the battle against colorectal cancer (CRC). CD73, encoded by the gene 5'-Nucleotidase Ecto (NT5E), is a major enzyme that generates extracellular adenosine. However, whether CD73 affects cancer progression and immune response in CRC remains unclear. Here, the clinical significance of CD73 was assessed in human CRC specimens using immunohistochemistry and bioinformatic analyses. We demonstrated that CD73 is elevated in CRC tissues, particularly in those with metastasis, and correlates with poor prognosis. Gain- and loss-of-function experiments demonstrate that tumor CD73 supports tumor progression and impairs the viability and effector functions of CD8+ T cells. Targeting CD73 on CRC cells reduces their malignant phenotypes and improves the anti-cancer response of CD8+ T cells in the tumor microenvironment (TME). Moreover, the combination of CD73 blockade and PD-1 inhibitors exhibited enhanced anti-cancer effects when compared to a single-agent treatment. Thus, CD73 may be a promising target in the treatment of CRC.
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Adenosina , Neoplasias Colorrectales , Humanos , 5'-Nucleotidasa/genética , Linfocitos T CD8-positivos , Evasión Inmune , Neoplasias Colorrectales/patología , Microambiente TumoralRESUMEN
Triple-negative breast cancer (TNBC) presents a formidable challenge in oncology due to its aggressive phenotype and the immunosuppressive nature of its tumor microenvironment (TME). In this issue of the JCI, Zhu, Banerjee, and colleagues investigated the potential of targeting the OTU domain-containing protein 4 (OTUD4)/CD73 axis to mitigate immunosuppression in TNBC. They identified elevated CD73 expression as a hallmark of immunosuppression in TNBC. Notably, the CD73 expression was regulated by OTUD4-mediated posttranslational modifications. Using ST80, a pharmacologic inhibitor of OTUD4, the authors demonstrated the restoration of cytotoxic T cell function and enhanced efficacy of anti-PD-L1 therapy in preclinical models. These findings underscore the therapeutic potential of targeting the OTUD4/CD73 axis in TNBC.
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5'-Nucleotidasa , Procesamiento Proteico-Postraduccional , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Humanos , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Microambiente Tumoral/inmunología , Femenino , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , AnimalesRESUMEN
AIMS: Acute kidney injury (AKI) is a public health problem and represents a risk factor for cardiovascular diseases (CVD) and vascular damage. This study aimed to investigate the impact of AKI on purinergic components in mice aorta. MAIN METHODS: The kidney ischemia was achieved by the occlusion of the left kidney pedicle for 60 min, followed by reperfusion for 8 (IR8) and 15 (IR15) days. Renal function was assessed through biochemical assays, while gene expression levels were evaluated by RT-qPCR. KEY FINDINGS: Analyses of renal parameters showed renal remodeling through mass loss in the left kidney and hypertrophy of the right kidney in the IR15 group. Furthermore, after 15 days, local inflammation was evidenced in the aorta. Moreover, the aorta purinergic components were significantly impacted by the renal ischemia and reperfusion model, with increases in gene expression of the pro-inflammatory purinoceptors P2Y1, P2Y2, P2Y6, and P2X4, potentially contributing to the vessel inflammation. The expression of NTPDase2 and ecto-5'-nucleotidase were also significantly increased in the aorta of the same group. In addition, both ATP and AMP hydrolysis were significantly increased in the aorta from IR15 animals, driving the entire purinergic cascade to the production of the anti-inflammatory adenosine. SIGNIFICANCE: In short, this is the first time that inflammation of the aorta due to AKI was shown to have an impact on purinergic signaling components, with emphasis on the adenosinergic pathway. This seems to be closely implicated in the establishment of vascular inflammation in this model of AKI and deserves to be further investigated.
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Lesión Renal Aguda , Riñón , Daño por Reperfusión , Transducción de Señal , Animales , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Ratones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/etiología , Riñón/metabolismo , Riñón/irrigación sanguínea , Riñón/patología , Masculino , Aorta/metabolismo , Aorta/patología , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Ratones Endogámicos C57BL , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/genéticaRESUMEN
Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries.
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Adenosina Trifosfato , Apirasa , Movimiento Celular , Células Madre Embrionarias , Humanos , Adenosina Trifosfato/metabolismo , Animales , Ratones , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/citología , Apirasa/metabolismo , Receptores Purinérgicos/metabolismo , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Quimiotaxis , Antígenos CD/metabolismo , Antígenos CD/genética , Sangre Fetal/citología , Sangre Fetal/metabolismo , Adenosina/metabolismo , Transducción de SeñalRESUMEN
Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-ß signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.
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5'-Nucleotidasa , Proteolisis , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Ubiquitinación , Proteasas Ubiquitina-EspecíficasRESUMEN
BACKGROUND: Autoimmune uveitis is an inflammatory disease triggered by an aberrant immune response. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) are emerging as potential therapeutic agents for this condition. CD73, an ectoenzyme present on MSC-sEVs, is involved in mitigating inflammation by converting extracellular adenosine monophosphate into adenosine. We hypothesize that the inhibitory effect of MSC-sEVs on experimental autoimmune uveitis (EAU) could be partially attributed to the surface expression of CD73. METHODS: To investigate novel therapeutic approaches for autoimmune uveitis, we performed lentiviral transduction to overexpress CD73 on the surface of MSC-sEVs, yielding CD73-enriched MSC-sEVs (sEVs-CD73). Mice with interphotoreceptor retinoid-binding protein (IRBP)-induced EAU were grouped randomly and treated with 50 µg MSC-sEVs, vector infected MSC-sEVs, sEVs-CD73 or PBS via single tail vein injection. We evaluated the clinical and histological features of the induced mice and analyzed the proportion and functional capabilities of T helper cells. Furthermore, T-cells were co-cultured with various MSC-sEVs in vitro, and we quantified the resulting inflammatory response to assess the potential therapeutic benefits of sEVs-CD73. RESULTS: Compared to MSC-sEVs, sEVs-CD73 significantly alleviates EAU, leading to reduced inflammation and diminished tissue damage. Treatment with sEVs-CD73 results in a decreased proportion of Th1 cells in the spleen, draining lymph nodes, and eyes, accompanied by an increased proportion of regulatory T-cells (Treg cells). In vitro assays further reveal that sEVs-CD73 inhibits T-cell proliferation, suppresses Th1 cells differentiation, and enhances Treg cells proportion. CONCLUSION: Over-expression of CD73 on MSC-sEVs enhances their immunosuppressive effects in EAU, indicating that sEVs-CD73 has the potential as an efficient immunotherapeutic agent for autoimmune uveitis.
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5'-Nucleotidasa , Enfermedades Autoinmunes , Vesículas Extracelulares , Células Madre Mesenquimatosas , Uveítis , Animales , Uveítis/patología , Uveítis/terapia , Uveítis/metabolismo , Uveítis/inmunología , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Ratones , Enfermedades Autoinmunes/terapia , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/inmunología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Femenino , Proteínas de Unión al Retinol , HumanosRESUMEN
The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.
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5'-Nucleotidasa , Adenosina , Proliferación Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Transducción de Señal , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Animales , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Adenosina/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Ratones , Humanos , Adenosina Monofosfato/metabolismo , Ratones Endogámicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/genéticaRESUMEN
Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment.
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5'-Nucleotidasa , Resistencia a Antineoplásicos , Melanoma , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf , Vemurafenib , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Humanos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Vemurafenib/farmacología , Oximas/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/genéticaRESUMEN
Immune escape is the main reason that immunotherapy is ineffective in hepatocellular carcinoma (HCC). Here, this study illustrates a pathway mediated by neutrophil extracellular traps (NETs) that can promote immune escape of HCC. Mechanistically, we demonstrated that NETs up-regulated CD73 expression through activating Notch2 mediated nuclear factor kappa B (NF-κB) pathway, promoting regulatory T cells (Tregs) infiltration to mediate immune escape of HCC. In addition, we found the similar results in mouse HCC models by hydrodynamic plasmid transfection. The treatment of deoxyribonuclease I (DNase I) could inhibit the action of NETs and improve the therapeutic effect of anti-programmed cell death protein 1 (PD-1). In summary, our results revealed that targeting of NETs was a promising treatment to improve the therapeutic effect of anti-PD-1.