RESUMEN
While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.
Asunto(s)
Antígeno CD47 , Macrófagos , Metástasis de la Neoplasia , Fagocitosis , Proteínas Proto-Oncogénicas c-myc , Escape del Tumor , Humanos , Masculino , Proteínas Portadoras , Antígeno CD47/metabolismo , Macrófagos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Transducción de Señal , Escape del Tumor/genética , Escape del Tumor/inmunología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/inmunología , Células Tumorales CultivadasRESUMEN
Regulatory T cells (Tregs) are essential for the inhibition of immunity and the maintenance of tissue homeostasis. Signals from the T-cell antigen receptor (TCR) are critical for early Treg development, their expansion, and inhibitory activity. Although TCR-engaged activation of the paracaspase MALT1 is important for these Treg activities, the MALT1 effector pathways in Tregs remain ill-defined. Here, we demonstrate that MALT1 protease activity controls the TCR-induced upregulation of the transcription factor MYC and the subsequent expression of MYC target genes in Tregs. These mechanisms are important for Treg-intrinsic mitochondrial function, optimal respiratory capacity, and homeostatic Treg proliferation. Consistently, conditional deletion of Myc in Tregs results similar to MALT1 inactivation in a lethal autoimmune inflammatory syndrome. Together, these results identify a MALT1 protease-mediated link between TCR signaling in Tregs and MYC control that coordinates metabolism and Treg expansion for the maintenance of immune homeostasis.
Asunto(s)
Proliferación Celular , Activación de Linfocitos , Mitocondrias/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Linfocitos T Reguladores/inmunología , Animales , Ratones , Ratones Transgénicos , Mitocondrias/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks definite treatment targets. Tumor immune microenvironment (TIME) heterogeneity has a profound impact on the immunotherapy response. Tumors with non-inflamed TIME derive limited benefit from immunotherapy. However, what drives the formation of the non-inflamed TIME in TNBC remains unclear. METHODS: Using our multiomics database of TNBC, we conducted an analysis to explore the key genomic events driving the formation of the non-inflamed TIME in TNBC. In vitro and in vivo studies further revealed potential mechanisms and the efficacy of combination treatment with immunotherapy. RESULTS: With transcriptomic and genomic data, we systematically analyzed the TIME of TNBC and revealed that the classical basal-like subtype of TNBC consisted of two distinct microenvironment phenotypes, defined as the 'inflamed' and 'non-inflamed' subtypes. We performed further screening and demonstrated that MYC amplification and overexpression led to low immune infiltration and cytolytic activity in TIME. Mechanistically, MYC bound to DNMT1 promoter and activated DNMT1 transcription in TNBC cells, thus suppressing the Cyclic GMP-AMP synthase (cGAS)-STING pathway via an epigenetic regulatory way. In MYC-overexpressing TNBC, decitabine, an Food and Drug Administration (FDA)-approved DNA methyltransferase inhibitor, converted tumors from non-inflamed to inflamed tumors by enhancing T cell infiltration. Furthermore, the combination of decitabine with programmed cell death protein 1 (PD-1) inhibitor reversed T cell exhaustion and improved T cell function in mouse models, which elicited potent antitumor activity in MYC-overexpressing TNBC. CONCLUSIONS: Our work elucidates that the classic oncogene MYC induces immune evasion by repressing innate immunity. Furthermore, we provide a rationale for combining DNA methyltransferase inhibition with immunotherapy for the treatment of MYC-overexpressing TNBC.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/inmunología , Inmunidad Innata/inmunología , Proteínas de la Membrana/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Animales , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Femenino , Xenoinjertos , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Escape del Tumor , Regulación hacia ArribaRESUMEN
In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations, or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE ) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40× magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology, and the slides can be stored for a long time.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , Hibridación in Situ/métodos , Proteínas Proto-Oncogénicas c-myc/inmunología , Compuestos Cromogénicos/química , ADN/genética , Sondas de ADN , Amplificación de Genes , Genes myc/genética , Genes myc/fisiología , Humanos , Inmunohistoquímica/métodos , Neoplasias , Adhesión en Parafina/métodos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Translocación GenéticaRESUMEN
Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.
Asunto(s)
Aminohidrolasas/genética , Antígeno B7-H1/genética , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Enzimas Multifuncionales/genética , Neoplasias Pancreáticas/genética , Procesamiento Proteico-Postraduccional , Linfocitos T Citotóxicos/inmunología , Aminohidrolasas/antagonistas & inhibidores , Aminohidrolasas/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Carcinogénesis/inmunología , Carcinogénesis/patología , Línea Celular Tumoral , Embrión de Mamíferos , Fibroblastos/inmunología , Fibroblastos/patología , Ácido Fólico/inmunología , Ácido Fólico/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/inmunología , Metilenotetrahidrofolato Deshidrogenasa (NADP)/antagonistas & inhibidores , Metilenotetrahidrofolato Deshidrogenasa (NADP)/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Enzimas Multifuncionales/antagonistas & inhibidores , Enzimas Multifuncionales/inmunología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Transducción de Señal , Linfocitos T Citotóxicos/patología , Carga Tumoral , Escape del Tumor , Uridina Difosfato N-Acetilglucosamina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Carcinoma de Células Renales/inmunología , Regulación Neoplásica de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Proteínas de Punto de Control Inmunitario/inmunología , Neoplasias Renales/inmunología , Tumor Rabdoide/inmunología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/mortalidad , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/inmunología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas de Punto de Control Inmunitario/genética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/mortalidad , Mutación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Estudios Retrospectivos , Tumor Rabdoide/tratamiento farmacológico , Tumor Rabdoide/genética , Tumor Rabdoide/mortalidad , Transducción de Señal , Análisis de Supervivencia , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunología , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/inmunologíaRESUMEN
MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Linfocitos B/efectos de los fármacos , Antígenos B7/genética , Células Epiteliales/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Anticuerpos Biespecíficos/biosíntesis , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos B7/antagonistas & inhibidores , Antígenos B7/inmunología , Ingeniería Celular/métodos , Línea Celular Tumoral , Citarabina/farmacología , Células Epiteliales/inmunología , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunosupresores/farmacología , Inmunoterapia/métodos , Masculino , Terapia Molecular Dirigida/métodos , Plásmidos/química , Plásmidos/metabolismo , Próstata/inmunología , Próstata/patología , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , TransfecciónRESUMEN
OBJECTIVE: to determine the association between the expression of lipoprotein lipase (LPL) and c-MYC genes inperipheral blood cells of chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophedepending on the mutational status of IGHV genes. METHODS: Analysis was performed in the group of 69 CLL patients irradiated due to the Chornobyl NPP accident (58clean-up workers of 1986 year, 6 inhabitants of radionuclide contaminated areas, and 5 evacuees). The IGHV genemutational status was studied by polymerase chain reaction (PCR) followed by direct sequencing. LPL and c-MYCexpression was evaluated by Quantitative Real-time PCR. Data were analyzed with the SPSS software package, version 20.0. RESULTS: Relative LPL expression levels in CLL samples ranged from 0 to 1663.5 (mean 138.47 ± 30.69, median 26.1).A strong correlation between individual LPL expression levels and IGHV mutational status was found (r = 0.684;p < 0.0001). The average relative c-MYC expression level was 5.7 ± 0.87 (median 2.86; range 0-48.5). No association between c-MYC expression and IGHV mutational status was found. Among unmutated IGHV cases, a correlationbetween LPL and c-MYC gene expression levels was identified: r = 0.351; p = 0.013. CONCLUSIONS: Our data confirm the dominant concept that unmutated IGHV CLL cases are more sensitive to the actionof proliferative stimuli compared to mutated IGHV CLL cases. This is manifested by an increase in the expression ofa functionally significant LPL gene, is one for the strongest negative prognostic markers in CLL.
Asunto(s)
Accidente Nuclear de Chernóbil , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Leucemia Linfocítica Crónica de Células B/genética , Lipoproteína Lipasa/genética , Proteínas Proto-Oncogénicas c-myc/genética , Exposición a la Radiación/efectos adversos , Traumatismos por Radiación/genética , Anciano , Contaminantes Radiactivos del Aire/efectos adversos , Socorristas , Femenino , Contaminación Radiactiva de Alimentos , Regulación de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/efectos de la radiación , Lipoproteína Lipasa/inmunología , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Proteínas Proto-Oncogénicas c-myc/inmunología , Traumatismos por Radiación/etiología , Traumatismos por Radiación/inmunología , Traumatismos por Radiación/patología , Radioisótopos , Contaminantes Radiactivos del Suelo/efectos adversos , UcraniaRESUMEN
Effector memory (EM) CD8+ T cells expressing lower levels of IL-7R α (IL-7Rαlow) from healthy individuals are partly compromised in vitro, but the identity of these cells has remained unclear. In this study, we demonstrate that human IL-7Rαlow EM CD8+ T cells are naturally occurring anergic cells in vivo and impaired in proliferation and IL-2 production but competent in IFN-γ and TNF-α production, a state that can be restored by IL-2 stimulation. IL-7Rαlow EM CD8+ T cells show decreased expression of GATA3 and c-MYC and are defective in metabolic reprogramming toward glycolysis, a process required for the proliferation of T cells. However, IL-7Rαlow EM CD8+ T cells can proliferate with TCR stimulation in the presence of IL-2 and IL-15, suggesting that these cells can be restored to normality or increased activity by inflammatory conditions and may serve as a reservoir for functional immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Glucólisis/inmunología , Receptores de Interleucina-7/inmunología , Línea Celular Tumoral , Proliferación Celular/fisiología , Células Cultivadas , Factor de Transcripción GATA3/inmunología , Voluntarios Sanos , Humanos , Memoria Inmunológica/inmunología , Interleucina-15/inmunología , Células Jurkat , Proteínas Proto-Oncogénicas c-myc/inmunología , Transducción de Señal/inmunologíaRESUMEN
The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues. ACTB and GAPDH were shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes using c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.
Asunto(s)
Colesteatoma del Oído Medio/genética , Oído Medio/patología , Membrana Mucosa/patología , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Colesteatoma del Oído Medio/inmunología , Colesteatoma del Oído Medio/patología , Colesteatoma del Oído Medio/cirugía , Oído Medio/inmunología , Oído Medio/cirugía , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/inmunología , Membrana Mucosa/cirugía , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Estándares de Referencia , Reproducibilidad de los Resultados , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Adulto JovenRESUMEN
Macrophages are highly plastic cells, responding to diverse environmental stimuli to acquire different functional phenotypes. Signaling through MAPKs has been reported to regulate the differentiation of macrophages, but the role of ERK5 in IL-4-mediated M2 macrophage differentiation is still unclear. Here, we showed that the ERK5 signaling pathway plays a critical role in IL-4-induced M2 macrophage differentiation. Pharmacologic inhibition of MEK5, an upstream activator of ERK5, markedly reduced the expression of classical M2 markers, such as Arg-1, Ym-1, and Fizz-1, as well as the production of M2-related chemokines and cytokines, CCL22, CCL17, and IGF-1 in IL-4-stimulated macrophages. Moreover, pharmacologic inhibition of ERK5 also decreased the expression of several M2 markers induced by IL-4. In accordance, myeloid cell-specific Erk5 depletion (Erk5∆mye ), using LysMcre /Erk5f/f mice, confirmed the involvement of ERK5 in IL-4-induced M2 polarization. Mechanistically, the inhibition of ERK5 did not affect STAT3 or STAT6 phosphorylation, suggesting that ERK5 signaling regulates M2 differentiation in a STAT3 and STAT6-independent manner. However, genetic deficiency or pharmacologic inhibition of the MEK5/ERK5 pathway reduced the expression of c-Myc in IL-4-activated macrophages, which is a critical transcription factor involved in M2 differentiation. Our study thus suggests that the MEK5/ERK5 signaling pathway is crucial in IL-4-induced M2 macrophage differentiation through the induction of c-Myc expression.
Asunto(s)
Diferenciación Celular/inmunología , Interleucina-4/inmunología , MAP Quinasa Quinasa 5/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/inmunología , Proteína Quinasa 7 Activada por Mitógenos/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Diferenciación Celular/genética , Regulación de la Expresión Génica/inmunología , Interleucina-4/genética , MAP Quinasa Quinasa 5/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Quinasa 7 Activada por Mitógenos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/inmunologíaRESUMEN
Regulatory T cell (Treg) activation and expansion occur during neonatal life and inflammation to establish immunosuppression, yet the mechanisms governing these events are incompletely understood. We report that the transcriptional regulator c-Myc (Myc) controls immune homeostasis through regulation of Treg accumulation and functional activation. Myc activity is enriched in Tregs generated during neonatal life and responding to inflammation. Myc-deficient Tregs show defects in accumulation and ability to transition to an activated state. Consequently, loss of Myc in Tregs results in an early-onset autoimmune disorder accompanied by uncontrolled effector CD4+ and CD8+ T cell responses. Mechanistically, Myc regulates mitochondrial oxidative metabolism but is dispensable for fatty acid oxidation (FAO). Indeed, Treg-specific deletion of Cox10, which promotes oxidative phosphorylation, but not Cpt1a, the rate-limiting enzyme for FAO, results in impaired Treg function and maturation. Thus, Myc coordinates Treg accumulation, transitional activation, and metabolic programming to orchestrate immune homeostasis.
Asunto(s)
Ácidos Grasos/metabolismo , Terapia de Inmunosupresión , Inflamación/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Linfocitos T Reguladores/inmunología , Transferasas Alquil y Aril/inmunología , Animales , Animales Recién Nacidos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Citometría de Flujo , Homeostasis/inmunología , Inflamación/genética , Proteínas de la Membrana/inmunología , Ratones , Oxidación-Reducción , Fosforilación Oxidativa , Proteínas Proto-Oncogénicas c-myc/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14. All displayed context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. In particular, clone 9E10, the most cited Myc-tag antibody, displayed high context-dependent detection variability, whereas others, notably 4A6 and 9B11, showed much less context sensitivity in their detection of Myc-tagged proteins. The very high context sensitivity of 9E10 was further substantiated by peptide microarray analyses. We conclude that recently developed, purpose-made monoclonal antibodies specific for Myc have much more uniform reactivity in diverse assays and are much less context sensitive than is the legacy antibody 9E10.
Asunto(s)
Anticuerpos Monoclonales , Epítopos , Proteínas Proto-Oncogénicas c-myc , Anticuerpos de Cadena Única , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Epítopos/química , Epítopos/inmunología , Humanos , Ratones , Células 3T3 NIH , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunologíaRESUMEN
BACKGROUND: The increase in CD4+ T cell infiltration and overproduction of CD4+ T cell-associated cytokines have been observed in the inflamed colon mucosa of patients with ulcerative colitis (UC); the underlying mechanisms are not fully understood. Survivin plays a critical role in the interference with apoptotic machinery. This study aims to elucidate the role of survivin in the interference with the apoptotic machinery in CD4+ T cells of UC patients. METHODS: Peripheral blood samples were collected from UC patients (UC group) and healthy subjects (healthy group). The apoptotic status in CD4+ T cells was analyzed by flow cytometry. RESULTS: We observed that the expression of survivin was significantly higher in CD4+ T cells of UC patients than in healthy subjects. UC CD4+ T cells were resistant to apoptosis induction. A complex of survivin and c-Myc, the transcription factor of FasL, was detected in CD4+ T cells in UC patients, which prevented the binding of c-Myc to the FasL promoter and interfered with the expression of FasL. Increased expression of survivin prevented the activation-induced CD4+ T cells from apoptosis. CONCLUSIONS: The data indicate that UC CD4+ T cells express high levels of survivin, which impairs the apoptotic machinery in CD4+ T cells and prevents the activation-induced CD4+ T cell apoptosis. Therefore, target therapy against survivin has translational potential in the treatment of UC patients.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Colitis Ulcerosa/inmunología , Survivin/inmunología , Adulto , Colitis Ulcerosa/patología , Proteína Ligando Fas/inmunología , Femenino , Humanos , Masculino , Proteínas Proto-Oncogénicas c-myc/inmunologíaRESUMEN
BACKGROUND: Burkitt leukemia (BL) with the precursor B-cell immunophenotype is a rarely reported condition. The prognosis of such patients is similar to that of classic BL. However, the combination of chromosomal translocations associated with bcl-2 and c-myc rearrangement has a poor prognosis. OBSERVATIONS: An 11-year-old child presented with fever and weakness. Bone marrow aspiration showed morphologically L1 type blasts and flow cytometry analysis was compatible with precursor B-cell immunophenotype. Cytogenetic analysis revealed a combination of t(8;14) and t(14;l8). CONCLUSIONS: The combination of t(8;14) and t(14;l8) can exhibit different immunophenotypical and morphologic features in leukemias.
Asunto(s)
Linfoma de Burkitt , Cromosomas Humanos/genética , Células Precursoras de Linfocitos B , Translocación Genética , Linfoma de Burkitt/sangre , Linfoma de Burkitt/genética , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Niño , Análisis Citogenético , Citometría de Flujo , Humanos , Inmunofenotipificación , Masculino , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunologíaRESUMEN
Diffuse large B-cell lymphoma-not otherwise specified (DLBCL-NOS) is a large and heterogeneous subgroup of non-Hodgkin lymphoma. DLBCL can be subdivided into germinal centre B-cell like (GCB) and activated B-cell like (ABC or non-GCB) using a gene-expression based or an immunohistochemical approach. In this study we aimed to identify additional proteins that are differentially expressed between GCB and non-GCB DLBCL. A reference super-SILAC mix, including proteins of eight B-cell lymphoma cell lines, was mixed with proteins isolated from seven non-GCB DLBCL and five GCB DLBCL patient tissue samples to quantify protein levels. Protein identification and quantification was performed by LC-MS. We identified a total of 4289 proteins, with a four-fold significant difference in expression between non-GCB and GCB DLBCL for 37 proteins. Four proteins were selected for validation in the same cases and replication in an independent cohort of 47 DLBCL patients by immunohistochemistry. In the validation cohort, we observed a non-significant trend towards the same differential expression pattern as observed in the proteomics. The replication study showed significant and consistent differences for two of the proteins: expression of glomulin (GLMN) was higher in GCB DLBCL, while expression of ribosomal protein L23 (RPL23) was higher in non-GCB DLBCL. These proteins are functionally linked to important pathways involving MYC, p53 and angiogenesis. In summary, we showed increased expression of RPL23 and decreased expression of GLMN in non-GCB compared to GCB DLBCL on purified primary DLBCL patient samples and replicated these results in an independent patient cohort.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Linfocitos B/inmunología , Biomarcadores de Tumor/genética , Centro Germinal/inmunología , Linfoma de Células B Grandes Difuso/diagnóstico , Proteómica/métodos , Proteínas Ribosómicas/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/patología , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Niño , Estudios de Cohortes , Femenino , Expresión Génica , Centro Germinal/patología , Humanos , Marcaje Isotópico/métodos , Activación de Linfocitos , Linfoma de Células B Grandes Difuso/clasificación , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Ribosómicas/inmunología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
BACKGROUND: C-MYC appears to initiate and maintain tumorigenesis through modulation of immune regulatory molecules such as PD-L1. The aim of our research was to evaluate the clinical implication of C-MYC expression in gastric adenocarcinoma in relation to the expression of the immune regulatory molecules PD-L1 and FOXP3. METHODS: Tissue samples were acquired from 182 cases of gastric adenocarcinoma that were surgically resected at Kyung Hee University Hospital at Gangdong from 2006 to 2012. Immunohistochemical staining for C-MYC, PD-L1, CD8 and FOXP3 was done. RESULTS: C-MYC overexpression showed a significant correlation with smaller tumor size, lower T category, lower N category, lower recurrence rate, and less lymphatic invasion. And C-MYC overexpression was negatively correlated with PD-L1 expression. The tumoral FOXP3 was positively correlated with C-MYC overexpression and Tregs count. PD-L1 expression was positively correlated with Tregs, CD8â¯+â¯T cells, and tumor infiltrating lymphocytes (TIL). Tregs count was positively correlated with CD8â¯+â¯T cells and TIL. CD8â¯+â¯T cells was positively correlated with TIL. CONCLUSION: We discovered that the immune regulatory effect of C-MYC and PD-L1, and the tumor suppressor function of tumoral FOXP3 had a significant influence on the tumor microenvironment (Tregs, CD8â¯+â¯T cells, and tumor infiltrating lymphocytes) in a complex manner. The C-MYC overexpression is a good prognostic factor in gastric adenocarcinoma.
Asunto(s)
Adenocarcinoma/patología , Biomarcadores de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Neoplasias Gástricas/patología , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/inmunología , Biomarcadores de Tumor/análisis , Supervivencia sin Enfermedad , Femenino , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/inmunología , Humanos , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-myc/inmunología , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Mst1 is a multifunctional serine/threonine kinase that is highly expressed in several immune organs. The role of Mst1 in the activation of dendritic cells (DCs), a key player of adaptive immunity, is poorly understood. In this study, we investigated the role of Mst1 in GM-CSF-induced bone marrow-derived DCs and the underlying mechanisms. Mst1-/- DCs in response to GM-CSF expressed higher levels of activation/maturation-related cell surface molecules, such as B7 and MHC class II than Mst1+/+ DCs. Furthermore, the expression of proinflammatory cytokines, such as IL-23, TNF-α, and IL-12p40, was increased in Mst1-/- DCs, indicating that Mst1-deficiency may induce the hyperactivation of DCs. Additionally, Mst1-/- DCs exhibited a stronger capacity to activate allogeneic T cells than Mst1+/+ DCs. Silencing of Mst1 in DCs promoted their hyperactivation, similar to the phenotypes of Mst1-/- DCs. Mst1-/- DCs exhibited an increase in Akt1 phosphorylation and c-myc protein levels. In addition, treatment with an Akt1 inhibitor downregulated the protein level of c-myc increased in Mst1-deficient DCs, indicating that Akt1 acts as an upstream inducer of the de novo synthesis of c-myc. Finally, Akt1 and c-myc inhibitors downregulated the increased expression of IL-23p19 observed in Mst1-knockdown DCs. Taken together, these data demonstrate that Mst1 negatively regulates the hyperactivation of DCs through downregulation of the Akt1/c-myc axis in response to GM-CSF, and suggest that Mst1 is one of the endogenous factors that determine the activation status of GM-CSF-stimulated inflammatory DCs.
Asunto(s)
Células Dendríticas/inmunología , Monocitos/inmunología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Transducción de Señal/inmunología , Animales , Células Dendríticas/patología , Ratones , Ratones Noqueados , Monocitos/patología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal/genéticaRESUMEN
cMyc is a characteristic oncogene with dual functions in cell proliferation and apoptosis. Since the overexpression of the cMyc protooncogene is a common event in the development and growth of various human types of cancer, the present study investigated whether oncogenic cMyc can alter natural killer (NK) cellmediated immunity through the expression of associated genes, using PCR, western blotting and flow cytometry assays. Furthermore, whether cMyc could influence the expression levels of natural killer group 2 member D (NKG2D) ligands, which are well known NK activation molecules, as well as NK cellmediated immunity, was investigated. cMyc was inhibited by 10058F4 treatment and small interfering RNA transfection. Upregulation of cMyc was achieved by transfection with a pCMV6myc vector. The inhibition of cMyc increased MHC class I polyeptiderelated sequence B and UL16 binding protein 1 expressions among NKG2D ligands, and the overexpression of cMyc suppressed the expression of all NKG2D ligands, except MHC class I polyeptiderelated sequence A. Furthermore, the alteration of cMyc activity altered the susceptibility of K562 cells to NK cells. These results suggested that the overexpression of cMyc may contribute to the immune escape of cancer cells and cell proliferation. Combined treatment with NKbased cancer immunotherapy and inhibition of cMyc may achieve improved therapeutic results.
Asunto(s)
Regulación Leucémica de la Expresión Génica/inmunología , Inmunidad Celular , Células Asesinas Naturales/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Escape del Tumor , Regulación hacia Arriba/inmunología , Humanos , Células K562 , Células Asesinas Naturales/patologíaRESUMEN
PURPOSE: The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status. EXPERIMENTAL DESIGN: A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses. RESULTS: Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as ARhigh risk) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. ARhigh risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000). CONCLUSIONS: We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. ARhigh risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients.