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BACKGROUND & AIMS: Low levels of toll-like receptor 3 (TLR3) in patients with hepatocellular carcinoma (HCC) are associated with poor prognosis, primarily owing to the loss of inflammatory signaling and subsequent lack of immune cell recruitment to the liver. Herein, we explore the role of TLR3-triggered apoptosis in HCC cells. METHODS: Quantitative reverse transcription PCR, western blotting, immunohistochemistry and comparative genomic hybridization were used to analyze human and mouse HCC cell lines, as well as surgically resected primary human HCCs, and to study the impact of TLR3 expression on patient outcomes. Functional analyses were performed in HCC cells, following the restoration of TLR3 by lentiviral transduction. The role of TLR3-triggered apoptosis in HCC was analyzed in vivo in a transgenic mouse model of HCC. RESULTS: Lower expression of TLR3 in tumor compared to non-tumor matched tissue was observed at both mRNA and protein levels in primary HCC, and was predictive of shorter recurrence-free survival after surgical resection in both univariate (hazard ratio [HR] 1.79; 95% CI 1.04-3.06; pâ¯=â¯0.03) and multivariate analyses (HR 1.73; CI 1.01-2.97; pâ¯=â¯0.04). Immunohistochemistry confirmed frequent downregulation of TLR3 in human and mouse primary HCC cells. None of the 6 human HCC cell lines analyzed expressed TLR3, and ectopic expression of TLR3 following lentiviral transduction not only restored the inflammatory response but also sensitized cells to TLR3-triggered apoptosis. Lastly, in the transgenic mouse model of HCC, absence of TLR3 expression was accompanied by a lower rate of preneoplastic hepatocyte apoptosis and accelerated hepatocarcinogenesis without altering the tumor immune infiltrate. CONCLUSION: Downregulation of TLR3 protects transforming hepatocytes from direct TLR3-triggered apoptosis, thereby contributing to hepatocarcinogenesis and poor patient outcome. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease associated with a poor prognosis. In patients with HCC, TLR3 downregulation is associated with reduced survival. Herein, we show that the absence of TLR3 is associated with a lower rate of apoptosis, and subsequently more rapid hepatocarcinogenesis, without any change to the immune infiltrate in the liver. Therefore, the poor prognosis associated with low TLR3 expression in HCC is likely linked to tumors ability to escape apoptosis. TLR3 may become a promising therapeutic target in TLR3-positive HCC.
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Carcinogénesis/metabolismo , Carcinoma Hepatocelular , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Pronóstico , Receptor Toll-Like 3/genética , Animales , Apoptosis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Femenino , Hepatectomía/métodos , Hepatectomía/mortalidad , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Ratones , Persona de Mediana Edad , Transducción de SeñalRESUMEN
BACKGROUND: Nanoparticles are increasingly suspected as a strong etiologic factor of granuloma formation. AIM OF THE STUDY: The aim of our study was to compare lung inflammatory response and histology changes following exposure of mice to two widely used nanoparticles: carbon nanotubes (MWCNT) and cadmium-based nanoparticles (QDOT705) in an attempt to better our understanding of granulomatous inflammation. MATERIALS AND METHODS: Various groups of mice were included: control mice and mice that were intranasally instilled with QDOT or MWCNT. At defined time points post-challenge, bronchoalveolar lavages (BALs) and lung tissues were collected to study inflammatory and histologic changes. RESULTS: Analyses of lung BAL fluids and tissues of nanoparticles-challenged mice in comparison to controls found: (1) increased cellularity in BALs, (2) increase of total protein concentration, LDH activity and proteolytic activity in BALs; (3) patchy granulomas, (4) macrophages, CD3 ± T, Treg and B cell infiltration in granulomatous areas; and (5) altered regulation of key inflammatory mediators and receptors. Importantly, these changes were nanoparticle type-dependent. CONCLUSION: Our work enhances understanding of nanoparticles-induced lung inflammatory and histological changes that result in granuloma formation. We provide compelling evidence that not only exposure to nanoparticles leads to granulomatous lung inflammation, but the severity of this latter is nanostructure type-dependent. Of importance, while nanotechnology has the potential to revolutionize various fields including medicine, nanoparticles form the potential for an entirely new lung health risk that it is necessary to take seriously into consideration by setting up and/or reinforcing adequate safety measures.
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Granuloma/patología , Nanopartículas/efectos adversos , Neumonía/inducido químicamente , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Cadmio/efectos adversos , Granuloma/etiología , Ratones , Nanopartículas/química , Nanotubos de Carbono/efectos adversos , Neumonía/etiologíaRESUMEN
TLR3 belongs to the family of intracellular TLRs that recognize nucleic acids. Endolysosomal localization and cleavage of intracellular TLRs play pivotal roles in signaling and represent fail-safe mechanisms to prevent self-nucleic acid recognition. Indeed, cleavage by cathepsins is required for native TLR3 to signal in response to dsRNA. Using novel Abs generated against TLR3, we show that the conserved loop exposed in LRR12 is the single cleavage site that lies between the two dsRNA binding sites required for TLR3 dimerization and signaling. Accordingly, we found that the cleavage does not dissociate the C- and N-terminal fragments, but it generates a very stable "cleaved/associated" TLR3 present in endolysosomes that recognizes dsRNA and signals. Moreover, comparison of wild-type, noncleavable, and C-terminal-only mutants of TLR3 demonstrates that efficient signaling requires cleavage of the LRR12 loop but not dissociation of the fragments. Thus, the proteolytic cleavage of TLR3 appears to fulfill function(s) other than separating the two fragments to generate a functional receptor.
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Transducción de Señal , Receptor Toll-Like 3/metabolismo , Sitios de Unión , Catepsinas/metabolismo , Línea Celular , Perfilación de la Expresión Génica , Aparato de Golgi/metabolismo , Humanos , Lisosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteolisis , Receptor Toll-Like 3/genéticaAsunto(s)
Autofagia , Secuenciación del Exoma , Sarcoidosis/genética , Serina-Treonina Quinasas TOR/genética , Exoma , Salud de la Familia , Femenino , Francia , Predisposición Genética a la Enfermedad , Variación Genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Virulencia , Proteína de Unión al GTP rac1/genéticaAsunto(s)
Alergia e Inmunología/historia , Células Dendríticas/fisiología , Inmunidad Celular , Monocitos/fisiología , Migración Transendotelial y Transepitelial , Animales , Presentación de Antígeno , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Historia del Siglo XX , Humanos , RatonesRESUMEN
The quest for targeted therapies is critical in the battle against cancer. The RAS/MAP kinase pathway is frequently implicated in neoplasia, with ERK playing a crucial role as the most distal kinase in the RAS signaling cascade. Our previous research demonstrated that the interaction between ERK and MYD88, an adaptor protein in innate immunity, is crucial for RAS-dependent transformation and cancer cell survival. In this study, we examine the biological consequences of disrupting the ERK-MYD88 interaction through the ERK D-recruitment site (DRS), while preserving ERK's kinase activity. Our results indicate that EI-52, a small-molecule benzimidazole targeting ERK-MYD88 interaction induces an HRI-mediated integrated stress response (ISR), resulting in immunogenic apoptosis specific to cancer cells. Additionally, EI-52 exhibits anti-tumor efficacy in patient-derived tumors and induces an anti-tumor T cell response in mice in vivo. These findings suggest that inhibiting the ERK-MYD88 interaction may be a promising therapeutic approach in cancer treatment.
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Bencimidazoles , Quinasas MAP Reguladas por Señal Extracelular , Factor 88 de Diferenciación Mieloide , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Humanos , Animales , Ratones , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Línea Celular Tumoral , Bencimidazoles/farmacología , Apoptosis/efectos de los fármacos , Muerte Celular Inmunogénica/efectos de los fármacos , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/metabolismo , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC(50) of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O'nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host-pathogen interactions and the rational design of vaccines against CHIKV.
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Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/virología , Anticuerpos Monoclonales/fisiología , Anticuerpos Neutralizantes/fisiología , Virus Chikungunya/inmunología , Pruebas de Neutralización/métodos , Proteínas del Envoltorio Viral/inmunología , Infecciones por Alphavirus/prevención & control , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Sitios de Unión de Anticuerpos , Línea Celular Transformada , Virus Chikungunya/patogenicidad , Chlorocebus aethiops , Células HEK293 , Humanos , Inmunización Pasiva/métodos , Inmunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo , Células VeroRESUMEN
The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.
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Glucosa , Hexosaminas , Receptores ErbB/genética , Glucosa/metabolismo , Glicosilación , Hexosaminas/metabolismo , Humanos , NutrientesRESUMEN
BACKGROUND: Chemokines and chemokine receptors are major actors of leukocytes trafficking and some have been shown to play an important role in cancer metastasis. Chemokines CCL19, CCL20 and CCL21 and their receptors CCR6 and CCR7, were assessed as potential biomarkers of metastatic dissemination in primary breast cancer. METHODS: Biomarker expression levels were evaluated using immunohistochemistry on paraffin-embedded tissue sections of breast cancer (n = 207). RESULTS: CCR6 was expressed by tumor cells in 35% of cases. CCR7 was expressed by spindle shaped stromal cells in 43% of cases but not by tumor cells in this series. CCL19 was the only chemokine found expressed in a significant number of breast cancers and was expressed by both tumor cells and dendritic cells (DC). CCR6, CCL19 and CCR7 expression correlated with histologic features of aggressive disease. CCR6 expression was associated with shorter relapse-free survival (RFS) in univariate and but not in multivariate analysis (p = 0.0316 and 0.055 respectively), and was not associated with shorter overall survival (OS). Expression of CCR7 was not significantly associated with shorter RFS or OS. The presence of CCL19-expressing DC was associated with shorter RFS in univariate and multivariate analysis (p = 0.042 and 0.020 respectively) but not with shorter OS. CONCLUSION: These results suggest a contribution of CCR6 expression on tumor cells and CCL19-expressing DC in breast cancer dissemination. In our series, unlike what was previously published, CCR7 was exclusively expressed on stromal cells and was not associated with survival.
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Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/fisiopatología , Regulación Neoplásica de la Expresión Génica , Ligandos , Receptores CCR6/metabolismo , Receptores CCR7/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Quimiocinas C/metabolismo , Femenino , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática/patología , Persona de Mediana Edad , Pronóstico , Células del Estroma/metabolismo , Células del Estroma/patología , Análisis de SupervivenciaRESUMEN
In cancer cells only, TLR3 acquires death receptor properties by efficiently triggering the extrinsic pathway of apoptosis with Caspase-8 as apical protease. Here, we demonstrate that in the absence of Caspase-8, activation of TLR3 can trigger a form of programmed cell death, which is distinct from classical apoptosis. When TLR3 was activated in the Caspase-8 negative neuroblastoma cell line SH-SY5Y, cell death was accompanied by lysosomal permeabilization. Despite caspases being activated, lysosomal permeabilization as well as cell death were not affected by blocking caspase-activity, positioning lysosomal membrane permeabilization (LMP) upstream of caspase activation. Taken together, our data suggest that LMP with its deadly consequences represents a "default" death mechanism in cancer cells, when Caspase-8 is absent and apoptosis cannot be induced.
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Apoptosis , Caspasa 8/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptor Toll-Like 3/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Interferón Tipo I/farmacología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Necroptosis/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Poli I-C/farmacologíaRESUMEN
INTRODUCTION: Most Toll-like receptors and IL-1/IL-18 receptors activate a signaling cascade via the adaptor molecule MyD88, resulting in NF-κB activation and inflammatory cytokine and chemokine production. Females are less susceptible than males to inflammatory conditions, presumably due to protection by estrogen. The exact mechanism underlying this protection is unknown. METHODS: MCF7 cells expressing wild-type or mutated LXXLL motif were used to determine MyD88/estrogen receptor (ER)-a interaction by immunoprecipitation and cell activation by ELISA and luciferase reporter assay. IL-1b and/or E2 were used to activate MCF7 cells expressing normal or knocked down levels of PRMT1. Finally, in situ proximity ligation assay with anti-MyD88 and anti-methylated ER-a (methER-a) antibodies was used to evaluate MyD88/methylated ER-a interaction in THP1 cells and histological sections. RESULTS: We show that MyD88 interacts with a methylated, cytoplasmic form of estrogen receptor-alpha (methER-α). This interaction is required for NF-κB transcriptional activity and pro-inflammatory cytokine production, and is dissociated by estrogen. Importantly, we show a strong gender segregation in gametogenic reproductive organs, with MyD88/methER-α interactions found in testicular tissues and in ovarian tissues from menopausal women, but not in ovaries from women age 49 and less - suggesting a role for estrogen in disrupting this complex in situ. DISCUSSION: Collectively, our results indicate that the formation of MyD88/methER-α complexes during inflammatory signaling and their disruption by estrogen may represent a mechanism that contributes to gender bias in inflammatory responses.
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Toll-like receptor 3 (TLR3) is an immune receptor that behaves like a death receptor in tumor cells, thereby providing an original target for cancer therapy. The therapeutic potential of TLR3 targeting in malignant mesothelioma, an aggressive and incurable neoplasia of the pleura and peritoneum, has so far not been addressed. We investigated TLR3 expression and sensitivity of human mesothelioma cell lines to the synthetic dsRNA Poly(I:C), alone or in combination with cisplatin, the gold standard chemotherapy in mesothelioma. Activation of TLR3 by Poly(I:C) induced apoptosis of 4/8 TLR3-positive cell lines but not of TLR3-negative cell lines. The combined cisplatin/Poly(I:C) treatment enhanced apoptosis of 3/4 Poly(I:C)-sensitive cell lines and overcame resistance to Poly(I:C) or cisplatin alone in 2/4 cell lines. Efficacy of the combined treatment relied on cisplatin-induced downregulation of c-FLIP, the main regulator of the extrinsic apoptotic pathway, leading to an enhanced caspase-8-mediated pathway. Of note, 6/6 primary cell samples isolated from patients with peritoneal mesothelioma expressed TLR3. Patient-derived cells were sensitive to Poly(I:C) alone while the combined cisplatin/Poly(I:C) treatment induced dramatic cell death. Our findings demonstrate that TLR3 targeting in combination with cisplatin presents an innovative therapeutic strategy in mesothelioma.
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Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Receptor Toll-Like 3/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Caspasa 8/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Mesotelioma/genética , Mesotelioma/fisiopatología , Mesotelioma Maligno , Poli I-C/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Recent studies have demonstrated that human dental pulp cells sense pathogens and elicit innate and/or adaptive immunity. Particular attention has been paid to odontoblasts that are situated at the pulp-dentin interface and constitute the first line of defense to cariogenic bacteria entering dentin after enamel disruption. In this review, recent in vitro and in vivo data suggesting that odontoblasts initiate immune/inflammatory events within the dental pulp in response to cariogenic bacteria are discussed. These data include sensing of pathogens by Toll-like receptors (TLRs), production of chemokines upon cell stimulation with microbial by-products and induction of dendritic cell migration. Additional results presented here reveal that all TLR genes are expressed in the healthy human dental pulp that is thus well equipped to combat pathogens entering the tissue. Seventeen chemokine genes including CXCL12, CCL2, CXCL9, CX3CL1, CCL8, CXCL10, CCL16, CCL5, CXCL2, CCL4, CXCL11 and CCL3, and 9 chemokine receptor genes including CXCR4, CCR1, CCR5, CX3CR1, CCR10 and CXCR3, are also expressed in pulp. TLR2, CCL2 and CXCL1 are upregulated in odontoblasts both under caries lesions and upon stimulation with pathogen by-products. These molecules thus appear as preferential targets for the design of therapeutic agents able to reduce the immune/inflammatory response to cariogenic bacteria and favor pulp healing.
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Pulpa Dental/inmunología , Odontoblastos/inmunología , Infecciones Bacterianas/inmunología , Quimiocinas/genética , Quimiocinas/fisiología , Secuencia Conservada , Caries Dental/inmunología , Caries Dental/patología , Humanos , Lipopolisacáridos/farmacología , Diente Molar/inmunología , Diente Molar/patología , Odontoblastos/efectos de los fármacos , Odontoblastos/microbiología , Receptores de Quimiocina/genética , Ácidos Teicoicos/farmacología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Enfermedades Dentales/inmunologíaRESUMEN
Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.
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Antígenos de Superficie/genética , Antígenos de Superficie/fisiología , Islotes Pancreáticos/citología , Células de Langerhans/citología , Lectinas Tipo C/genética , Lectinas Tipo C/fisiología , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/fisiología , 9,10-Dimetil-1,2-benzantraceno , Animales , Antígenos/metabolismo , Blastocisto/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Carcinógenos , Movimiento Celular , Fenómenos Fisiológicos Celulares , Gránulos Citoplasmáticos/metabolismo , Células Dendríticas , Relación Dosis-Respuesta a Droga , Electroporación , Embrión de Mamíferos/citología , Citometría de Flujo , Vectores Genéticos , Inmunohistoquímica , Islotes Pancreáticos/fisiología , Cinética , Lectinas/metabolismo , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Modelos Genéticos , Mutagénesis , Mutación , Neoplasias/inducido químicamente , Ovalbúmina/metabolismo , Fenotipo , Células Madre/citologíaRESUMEN
PURPOSE: The immunomodulatory properties of Toll-like receptors (TLR) agonists have inspired their use as experimental adjuvants for vaccination of cancer patients. However, it is now well recognized that TLR expression is not restricted to immune cells but can also be found in many cell types, including those giving rise to tumors. It is therefore mandatory to explore the potential effects of TLR triggering directly on tumor cells. EXPERIMENTAL DESIGN: In the present work, we have investigated TLR3 protein expression in melanoma cell lines derived from patients, and analyzed the effects of TLR3 agonists on tumor cell survival. Moreover, we used RNA interference to stably knock down TLR3 expression and study the involvement of this receptor in dsRNA-induced effects on melanoma cells viability. RESULTS: Human melanoma cells can express functional TLR3 protein. Interestingly, the engagement of the receptor by TLR3 agonists can directly inhibit cell proliferation and induce tumor cell death when combined to treatment with either type I IFN or protein synthesis inhibitors. These effects were shown by RNA interference to be largely dependent on TLR3. Moreover, TLR3-mediated cell death involves the activation of caspases and engages both extrinsic and intrinsic apoptotic pathways. CONCLUSION: TLR3 protein can be expressed in human melanoma cells, where it can deliver proapoptotic and antiproliferative signaling. Altogether, these results suggest that TLR3 agonists represent very promising adjuvants for cancer vaccines not only based on their well-described immunostimulatory properties, but also due to their newly identified cytostatic and cytotoxic effects directly on tumor cells.
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Apoptosis/inmunología , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Receptor Toll-Like 3/metabolismo , Caspasas/metabolismo , Proliferación Celular , Humanos , Inmunoprecipitación , Interferón-alfa/farmacología , Lentivirus/genética , Melanoma/patología , Melanoma/terapia , Poli I-C/farmacología , Interferencia de ARN , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Receptor Toll-Like 3/agonistasRESUMEN
PURPOSE: In a previous immunohistochemical study of dendritic cells (DC) in sentinel lymph nodes (SLN) draining regressing melanomas, we found that the accumulation of mature DC-LAMP(+) DCs in SLNs was associated with local expansion of antigen-specific memory effector CTLs and the absence of metastasis in downstream lymph nodes. The aim of this study was to investigate the prognostic importance of the maximal density of mature DCs in SLNs. EXPERIMENTAL DESIGN: A total of 458 consecutive patients with micrometastatic melanoma within SLNs were eligible for analysis. The maximal density of mature DC-LAMP(+) DCs was evaluated by three independent observers and categorized into three classes (<100, 100 to <200, and >or=200/mm(2)). RESULTS: There was excellent interobserver reproducibility for maximum density of mature DC-LAMP(+) DC scores (kappa score = 0.82). There were differences in the maximal density scores and staining intensity according to the treating melanoma center (P < 0.001). The higher the mature DC density in the SLN is, the longer is the duration of survival [P = 0.047; hazard ratio, 0.70; 95% confidence interval, 0.50-1.00]. Adjusted by thickness and ulceration, the prognostic importance of DC density was lower (P = 0.36). CONCLUSION: This study is the first to report the prognostic value of DC-LAMP(+) DC counts in SLNs containing metastatic melanoma. Patients with a high density of mature DCs (>or=200/mm(2)) have the lowest risk of death. It also provides evidence that a lack of maturation in the SLNs is important in biological facilitation of melanoma progression.
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Células Dendríticas/citología , Proteínas de Membrana de los Lisosomas/biosíntesis , Melanoma/metabolismo , Biopsia del Ganglio Linfático Centinela , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Resultado del TratamientoRESUMEN
The sensitivity of cells to death receptor-induced apoptosis is commonly controlled by multiple checkpoints in order to limit induction of excessive or unnecessary death. Although cytotoxic in various cancer cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) does not trigger apoptosis in most non-transformed cells. The molecular nature of the checkpoints that normally protect the cells from TRAIL-induced death are not fully understood. Endoplasmic reticulum (ER) stress has been reported to switch the sensitivity of human cells to the cytotoxic effect of TRAIL, suggesting that this cellular state perturbs some of these protective mechanisms. We found that tunicamycin (TU), but no other ER stress inducers, sensitized mouse fibroblasts and hippocampal neuronal cells to TRAIL-induced apoptosis. Importantly, the sensitization was specific to TRAIL and not caused by differences in ER stress induction. Instead, it relied on the inhibition of N-glycosylation of the mouse TRAIL receptor (mTRAIL-R). Inhibition of N-glycosylation did not alter cell surface expression of mTRAIL-R but enhanced its ability to bind TRAIL, and facilitated mTRAIL-R oligomerization, which resulted in enhanced death-inducing signaling complex (DISC) formation and caspase-8 activation. Remarkably, reconstitution of mTRAIL-R-deficient cells with a version of mTRAIL-R mutated for the three N-glycosylation sites identified in its ectodomain confirmed higher sensitivity to TRAIL-induced apoptosis. Together, our results demonstrate that inhibition of N-glycosylation of mTRAIL-R, and not ER stress induction, sensitizes mouse cells to TRAIL-induced apoptosis. We therefore reveal a new mechanism restraining TRAIL cytotoxicity in mouse cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células 3T3 , Animales , Fibroblastos/metabolismo , Fibroblastos/patología , Glicosilación , Células HEK293 , Células HeLa , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Transducción de SeñalRESUMEN
Toll-like receptor 3 (TLR3) mediates innate immune responses by sensing viral dsRNA, but also induces apoptosis selectively in cancer cells. Our analysis by immunohistochemistry revealed that TLR3 is frequently overexpressed in 130 non-small cell lung cancer (NSCLC) patients' samples compared with normal bronchial epithelium (P < 0.0001, Mann-Whitney test), supporting the therapeutic potential of TLR3 ligand for this type of cancer. However, a proportion of TLR3-expressing cancer cell lines, including NSCLC, remain resistant to TLR3-mediated apoptosis, and the underlying mechanism of resistance remains unclear. We here investigated the molecular basis conferring resistance to non-transformed vs. transformed cells against TLR3-mediated cell death. In non-transformed epithelial cells cellular FLICE-like inhibitory protein (c-FLIP) and cellular Inhibitor of APoptosis (cIAPs) ubiquitin ligases exerted an efficient double brake on apoptosis signaling. In contrast, releasing only one of these two brakes was sufficient to overcome the resistance of 8/8 cancer cell lines tested. Remarkably, the release of the c-FLIP, but not cIAPs, brake only results in the sensitization of all human cancer cells to TLR3-mediated apoptosis. Taking advantage of the difference between transformed and non-transformed cells, we developed a rational strategy by combining the chemotherapeutic agent paclitaxel, which decreases c-FLIP expression, with TLR3 ligand. This combination was highly synergistic for triggering apoptosis in cancer cells but not in non-transformed cells. In vivo, the combination of paclitaxel with dsRNA delayed tumor growth and prolonged survival in a mouse xenograft lung tumor model. In conclusion, combining the release of the c-FLIP brake with TLR3 ligand synergizes to selectively kill cancer cells, and could represent an efficient and safe therapy against TLR3-expressing cancers such as NSCLC.
Asunto(s)
Apoptosis/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Receptor Toll-Like 3/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: Sarcoidosis (OMIM 181000) is a multi-systemic granulomatous disorder of unknown origin. Despite multiple genome-wide association (GWAS) studies, no major pathogenic pathways have been identified to date. To find out relevant sarcoidosis predisposing genes, we searched for de novo and recessive mutations in 3 young probands with sarcoidosis and their healthy parents using a whole-exome sequencing (WES) methodology. METHODS: From the SARCFAM project based on a national network collecting familial cases of sarcoidosis, we selected three families (trios) in which a child, despite healthy parents, develop the disease before age 15 yr. Each trio was genotyped by WES (Illumina HiSEQ 2500) and we selected the gene variants segregating as 1) new mutations only occurring in affected children and 2) as recessive traits transmitted from each parents. The identified coding variants were compared between the three families. Allelic frequencies and in silico functional results were analyzed using ExAC, SIFT and Polyphenv2 databases. The clinical and genetic studies were registered by the ClinicalTrials.gov - Protocol Registration and Results System (PRS) ( https://clinicaltrials.gov ) receipt under the reference NCT02829853 and has been approved by the ethical committee (CPP LYON SUD EST - 2 - REF IRB 00009118 - September 21, 2016). RESULTS: We identified 37 genes sharing coding variants occurring either as recessive mutations in at least 2 trios or de novo mutations in one of the three affected children. The genes were classified according to their potential roles in immunity related pathways: 9 to autophagy and intracellular trafficking, 6 to G-proteins regulation, 4 to T-cell activation, 4 to cell cycle and immune synapse, 2 to innate immunity. Ten of the 37 genes were studied in a bibliographic way to evaluate the functional link with sarcoidosis. CONCLUSIONS: Whole exome analysis of case-parent trios is useful for the identification of genes predisposing to complex genetic diseases as sarcoidosis. Our data identified 37 genes that could be putatively linked to a pediatric form of sarcoidosis in three trios. Our in-depth focus on 10 of these 37 genes may suggest that the formation of the characteristic lesion in sarcoidosis, granuloma, results from combined deficits in autophagy and intracellular trafficking (ex: Sec16A, AP5B1 and RREB1), G-proteins regulation (ex: OBSCN, CTTND2 and DNAH11), T-cell activation (ex: IDO2, IGSF3), mitosis and/or immune synapse (ex: SPICE1 and KNL1). The significance of these findings needs to be confirmed by functional tests on selected gene variants.