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1.
Mol Med Rep ; 20(4): 3301-3307, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432134

RESUMO

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.


Assuntos
Regulação Leucêmica da Expressão Gênica/imunologia , Imunidade Celular , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Evasão Tumoral , Regulação para Cima/imunologia , Humanos , Células K562 , Células Matadoras Naturais/patologia
2.
Nat Commun ; 10(1): 1911, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015454

RESUMO

Multiple myeloma is a malignancy of antibody-secreting plasma cells. Most patients benefit from current therapies, however, 20% of patients relapse or die within two years and are deemed high risk. Here we analyze structural variants from 795 newly-diagnosed patients as part of the CoMMpass study. We report translocations involving the immunoglobulin lambda (IgL) locus are present in 10% of patients, and indicative of poor prognosis. This is particularly true for IgL-MYC translocations, which coincide with focal amplifications of enhancers at both loci. Importantly, 78% of IgL-MYC translocations co-occur with hyperdiploid disease, a marker of standard risk, suggesting that IgL-MYC-translocated myeloma is being misclassified. Patients with IgL-translocations fail to benefit from IMiDs, which target IKZF1, a transcription factor that binds the IgL enhancer at some of the highest levels in the myeloma epigenome. These data implicate IgL translocation as a driver of poor prognosis which may be due to IMiD resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Cadeias lambda de Imunoglobulina/genética , Mieloma Múltiplo/diagnóstico , Proteínas do Mieloma/genética , Translocação Genética , Antineoplásicos/uso terapêutico , Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos/imunologia , Elementos Facilitadores Genéticos , Loci Gênicos , Genoma Humano , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/imunologia , Fatores Imunológicos/uso terapêutico , Lenalidomida/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Plasmócitos/imunologia , Plasmócitos/patologia , Prognóstico , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Recidiva , Análise de Sobrevida , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Sequenciamento Completo do Genoma
4.
Front Immunol ; 9: 2715, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524445

RESUMO

Appropriate PI3K signals generated by the antigen receptor are essential to promote B cell development. Regulation of recombination activating gene (RAG)-1 and RAG-2 expression is one key process that is mediated by PI3K to ensure developmental progression and selection. When PI3K signals are too high or too low, expression of RAGs does not turn off and B cell development is impaired or blocked. Yet, the mechanism which tunes PI3K activity to control RAG expression during B cell development in the bone marrow is unknown. Recently we showed that a c-Myc/miR17-92/PTEN axis regulates PI3K activity for positive and negative selection of immature B cells. Here, we show that the c-Myc/miR17-92/PTEN axis tunes PI3K activity to control the expression of RAGs in proB cells. Using different genetically engineered mouse models we show that impaired function of the c-Myc/miR17-92/PTEN axis alters the PI3K/Akt/Foxo1 pathway to result in dis-regulated expression of RAG and a block in B cell development. Studies using 38c-13 B lymphoma cells, where RAGs are constitutively expressed, suggest that this regulatory effect is mediated post-translationally through Foxo1.


Assuntos
Regulação da Expressão Gênica/imunologia , Rearranjo Gênico do Linfócito B , MicroRNAs/imunologia , PTEN Fosfo-Hidrolase/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Células Precursoras de Linfócitos B/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Recombinação Genética/imunologia , Animais , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Células Precursoras de Linfócitos B/citologia , Proteínas Proto-Oncogênicas c-myc/genética
5.
J Autoimmun ; 94: 90-98, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30077426

RESUMO

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.


Assuntos
Artrite Juvenil/imunologia , Artrite Reumatoide/imunologia , Autofagia/imunologia , Redes Reguladoras de Genes/imunologia , Memória Imunológica , Proteínas Proto-Oncogênicas c-myc/genética , Adolescente , Adulto , Animais , Artrite Juvenil/genética , Artrite Juvenil/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Autofagia/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Estudos de Casos e Controles , Metilação de DNA , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos DBA , Oxidiazóis/farmacologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia
6.
Biochem Biophys Res Commun ; 503(4): 2807-2813, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30103947

RESUMO

The regulation of natural killer (NK) cell activity is an important research goal for the development of immunotherapies. In this study, we identified transcription factors affecting NK cell activity. In particular, we screened transcription factors affected by interleukin-2 (IL-2) and transforming growth factor-beta (TGF-ß) by protein/DNA arrays using primary NK cells. We found that celastrol, a c-Myb inhibitor, inhibited NK-92 cells more strongly than any other inhibitors of transcription factor candidates. In addition, c-Myb and c-Myb-related signaling molecules, e.g., Nemo-like kinase (NLK) and c-Myc, were regulated by the activation status of NK cells, suggesting that c-Myb is a key regulator of NK cell activity. We also found that celastrol inhibits NK-92-cell-mediated cytotoxicity via the downregulation of NKG2D and granzyme B. Knockdown studies also showed that c-Myb is important for NK cell activation. In particular, the knockdown of c-Myb did not significantly affect NK cell proliferation and survival but decreased the secretion of IFN-γ and the cytotoxicity of NK cells. Our data demonstrate that c-Myb plays a critical role in the activation of NK cells and therefore is a therapeutic target for cancer and viral diseases.


Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myb/genética , Triterpenos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Granzimas/genética , Granzimas/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas c-myb/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myb/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia
7.
J Immunol ; 201(5): 1452-1459, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30012846

RESUMO

γδ NKT cells are neonatal-derived γδ T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of γδ NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote γδ NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating γδ NKT cell expansion. We observed a higher expression of Egr family genes in γδ NKT cells compared with the conventional γδ T cell population. Loss of function of Id3 caused an expansion of γδ NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient γδ NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated γδ NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient γδ NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2-double-deficient γδ NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2-Id3-E2A, which normally restricts the population size of γδ NKT cells by adjusting Egr2 dosage and c-Myc expression.


Assuntos
Proliferação de Células/fisiologia , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas Inibidoras de Diferenciação/imunologia , Células T Matadoras Naturais/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas Inibidoras de Diferenciação/genética , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Receptores de Antígenos de Linfócitos T gama-delta
8.
FASEB J ; 32(10): 5312-5325, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29718706

RESUMO

A key event required for effective resolution of inflammation is efferocytosis, which is defined as phagocytic removal of apoptotic cells mostly by macrophages acquiring an alternatively activated phenotype (M2). c-Myc has been reported to play a role in alternative activation of human macrophages and is proposed as one of the M2 macrophage markers. We found that M2-like peritoneal macrophages from zymosan A-treated mice exhibited a marked accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, ectopic expression of Myc-nick in murine bone marrow-derived macrophages promoted the M2 polarization and, consequently, enhanced their efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be tightly associated with α-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5) activity. These findings suggest Myc-nick as a novel proresolving mediator that has a fundamental function in maintaining homeostasis under inflammatory conditions.-Zhong, X., Lee, H.-N., Kim, S. H., Park, S.-A., Kim, W., Cha, Y.-N., Surh, Y.-J. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.


Assuntos
Células da Medula Óssea/imunologia , Macrófagos Peritoneais/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Acetilação , Animais , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/patologia , Histona Acetiltransferases/imunologia , Inflamação/imunologia , Inflamação/patologia , Macrófagos Peritoneais/patologia , Camundongos , Tubulina (Proteína)/imunologia , Fatores de Transcrição de p300-CBP/imunologia
9.
J Clin Invest ; 127(10): 3609-3623, 2017 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-28846070

RESUMO

During an immune response, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that "pre-effector" and "pre-memory" cells resulting from the first CD8+ T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8+ T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8+ T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Glicólise/imunologia , Memória Imunológica , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Camundongos Mutantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Proto-Oncogênicas c-myc/metabolismo
10.
Oncogene ; 36(45): 6235-6243, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-28714960

RESUMO

Non-small cell lung cancer (NSCLC) is one of the most common and malignant carcinoma worldwide, and the incidence and mortality are increasing rapidly. Immunotherapy targeting programmed death 1/programmed death ligand 1 (PD-L1) signaling has shown prominent clinical effects in treating NSCLC; however, a poor understanding of the associated regulating molecular mechanisms of PD-L1 has become one of the biggest obstacles for further improving efficacy. Bridging integrator-1 (BIN1) can regulate numerous cancer-related molecules to exert multiple tumor-suppressing effects by either interacting or not interacting with c-MYC. In the present study, we observed that there exists a negative correlation between the expression of PD-L1 and BIN1 in NSCLC tissues. The expression levels of BIN1 and PD-L1 were significantly related to the tumor, lymph node and metastasis grade (TNM) stage, invasion range and lymph node metastasis. Simultaneously, for NSCLC patients, the expression statuses of BIN1 and PD-L1 might be independent prognostic factors. Furthermore, the expression of tumor-infiltrating lymphocytes was positively associated with BIN1 expression and negatively related to PD-L1 expression in NSCLC tissues. Importantly, we showed that PD-L1 was under the control of BIN1. In addition, the overexpression of BIN1 could inhibit the c-MYC and epithelial growth factor receptor (EGFR)-dependent PD-L1 expression and reverse the suppressive immuno-microenvironment in vivo. Taken together, our findings indicated that BIN1 restoration in NSCLC could reverse PD-L1-mediated immune escape by inactivating the c-MYC and EGFR/mitogen-activated protein kinase pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Receptores ErbB/imunologia , Neoplasias Pulmonares/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Nucleares/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Genes myc , Xenoenxertos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/imunologia , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Transfecção , Evasão Tumoral
11.
J Leukoc Biol ; 102(4): 953-964, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28663244

RESUMO

Leukocyte extravasation is a crucial feature of the normal immune response to disease and infection and is implicated in various pathologies during chronic inflammatory disease. P-Selectin glycoprotein ligand-1 (PSGL-1) is critical for leukocyte extravasation; however, despite extensive study, it remains unclear how its expression is regulated, which in turn, impedes a more precise understanding of how its expression level affects transmigration. To investigate the regulation of PSGL-1, 60 subjects, with or without HIV infection, were recruited and PSGL-1 expression in monocytes was measured. PSGL-1 was found to be up-regulated on leukocytes from HIV-infected individuals, and the physiologically relevant mediators soluble CD40 ligand (sCD40L) and glutamate were able to induce PSGL-1 transcription in human monocytes ex vivo. HIV-1 induced PSGL-1 induction, and its dependence on CD40L was validated further by use of the mouse-tropic HIV (EcoHIV) mouse model of HIV infection in C57BL/6 and CD40L knockout (KO) mice. To investigate crosstalk between the signaling cascades induced by CD40L and glutamate that lead to PSGL-1 induction, a network-based, discrete dynamic model was developed. The model reveals the MAPK pathway and oxidative stress as critical mediators of crosstalk between CD40L and glutamate-induced pathways. Importantly, the model predicted induction of the c-Myc transcription factor upon cotreatment, which was validated using transcriptomic data and pharmacologic inhibition of c-Myc. This study suggests a novel systems serology approach for translational research and reveals a mechanism for PSGL-1 transcriptional regulation, which might be leveraged to identify novel targets for therapeutic intervention.


Assuntos
Regulação da Expressão Gênica/imunologia , Infecções por HIV/imunologia , Glicoproteínas de Membrana/imunologia , Modelos Biológicos , Proteínas Proto-Oncogênicas c-myc/imunologia , Transdução de Sinais/imunologia , Adulto , Idoso , Animais , Feminino , HIV-1/imunologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade
12.
Anal Biochem ; 532: 38-44, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600127

RESUMO

A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.


Assuntos
Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/análise , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Recombinantes de Fusão/análise , Anticorpos de Cadeia Única/análise , Ciclização , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
13.
PLoS One ; 12(6): e0178986, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28575129

RESUMO

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.


Assuntos
Ligamento Amarelo/citologia , Ligamento Amarelo/patologia , Ossificação Heterotópica/patologia , Osteoblastos/patologia , Fator de Necrose Tumoral alfa/imunologia , Proliferação de Células , Células Cultivadas , Ciclina D1/imunologia , Humanos , Ligamento Amarelo/imunologia , Sistema de Sinalização das MAP Quinases , Ossificação Heterotópica/imunologia , Osteoblastos/imunologia , Osteogênese , Proteínas Proto-Oncogênicas c-myc/imunologia , Fase S , Vértebras Torácicas/imunologia , Vértebras Torácicas/patologia
14.
Cell Metab ; 25(6): 1282-1293.e7, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28416194

RESUMO

Immune cells function in diverse metabolic environments. Tissues with low glucose and high lactate concentrations, such as the intestinal tract or ischemic tissues, frequently require immune responses to be more pro-tolerant, avoiding unwanted reactions against self-antigens or commensal bacteria. T-regulatory cells (Tregs) maintain peripheral tolerance, but how Tregs function in low-glucose, lactate-rich environments is unknown. We report that the Treg transcription factor Foxp3 reprograms T cell metabolism by suppressing Myc and glycolysis, enhancing oxidative phosphorylation, and increasing nicotinamide adenine dinucleotide oxidation. These adaptations allow Tregs a metabolic advantage in low-glucose, lactate-rich environments; they resist lactate-mediated suppression of T cell function and proliferation. This metabolic phenotype may explain how Tregs promote peripheral immune tolerance during tissue injury but also how cancer cells evade immune destruction in the tumor microenvironment. Understanding Treg metabolism may therefore lead to novel approaches for selective immune modulation in cancer and autoimmune diseases.


Assuntos
Microambiente Celular/imunologia , Reprogramação Celular/imunologia , Fatores de Transcrição Forkhead/imunologia , Glucose/imunologia , Ácido Láctico/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular , Microambiente Celular/genética , Reprogramação Celular/genética , Fatores de Transcrição Forkhead/genética , Glucose/genética , Glicólise/genética , Glicólise/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia
15.
Clin Cancer Res ; 23(15): 4462-4472, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28270499

RESUMO

Purpose: This study sought to evaluate the expression of programmed cell death-ligand-1 (PD-L1) and HLA class I on neuroblastoma cells and programmed cell death-1 (PD-1) and lymphocyte activation gene 3 (LAG3) on tumor-infiltrating lymphocytes to better define patient risk stratification and understand whether this tumor may benefit from therapies targeting immune checkpoint molecules.Experimental Design:In situ IHC staining for PD-L1, HLA class I, PD-1, and LAG3 was assessed in 77 neuroblastoma specimens, previously characterized for tumor-infiltrating T-cell density and correlated with clinical outcome. Surface expression of PD-L1 was evaluated by flow cytometry and IHC in neuroblastoma cell lines and tumors genetically and/or pharmacologically inhibited for MYC and MYCN. A dataset of 477 human primary neuroblastomas from GEO and ArrayExpress databases was explored for PD-L1, MYC, and MYCN correlation.Results: Multivariate Cox regression analysis demonstrated that the combination of PD-L1 and HLA class I tumor cell density is a prognostic biomarker for predicting overall survival in neuroblastoma patients (P = 0.0448). MYC and MYCN control the expression of PD-L1 in neuroblastoma cells both in vitro and in vivo Consistently, abundance of PD-L1 transcript correlates with MYC expression in primary neuroblastoma.Conclusions: The combination of PD-L1 and HLA class I represents a novel prognostic biomarker for neuroblastoma. Pharmacologic inhibition of MYCN and MYC may be exploited to target PD-L1 and restore an efficient antitumor immunity in high-risk neuroblastoma. Clin Cancer Res; 23(15); 4462-72. ©2017 AACR.


Assuntos
Antígeno B7-H1/genética , Genes MHC Classe I/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/imunologia , Azepinas/administração & dosagem , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes MHC Classe I/imunologia , Humanos , Lactente , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Proteína Proto-Oncogênica N-Myc/imunologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/imunologia , Neuroblastoma/patologia , Prognóstico , Receptor de Morte Celular Programada 1/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Triazóis/administração & dosagem
16.
Nat Commun ; 8: 14581, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28262675

RESUMO

The Eµ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eµ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eµ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.


Assuntos
Linfócitos B/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/genética , Mutação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/genética , Alelos , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Sistemas CRISPR-Cas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Modelos Animais de Doenças , Edição de Genes , Frequência do Gene , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Repressoras/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transcriptoma , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Sequenciamento Completo do Genoma
17.
Am J Surg Pathol ; 41(4): 541-549, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28291124

RESUMO

Components of the B-cell receptor (BCR) signaling pathway represent promising therapeutic targets in diffuse large B-cell lymphoma (DLBCL) and other B-cell malignancies. MYC, a transcriptional factor and oncoprotein, is overexpressed in a fraction of DLBCL and indicates poor prognosis and aggressive clinical course when treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). However, BCR signaling status in MYC-positive DLBCL cases and the potential efficacy of BCR signal inhibitors in treating this aggressive disease are unknown. To further elucidate the BCR signaling pathway in MYC-positive DLBCL, we analyzed the levels of BCR-associated genes according to MYC gene status, detected phosphorylated protein with primary DLBCL samples, and estimated the patient survival with MYC expression. In addition, we manipulated MYC gene expression and tested its effects on BCR signaling in vitro. We found that CD19, SYK, and BLK were highly expressed in DLBCL with MYC gene overexpression. MYC-positive DLBCL had higher levels of pSYK and pBLK, but only pSYK level correlated with patient survival. The in vitro studies demonstrated that overexpression of the MYC gene augmented BCR signaling, whereas MYC gene knockdown attenuated BCR signaling. Thus, MYC protein-positive DLBCL features highly activated BCR signaling and may represent a potential candidate for BCR inhibitor therapy.


Assuntos
Linfócitos B/metabolismo , Biomarcadores Tumorais/metabolismo , Ativação Linfocitária , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Interferência de RNA , Receptores de Antígenos de Linfócitos B/imunologia , Fatores de Risco , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Fatores de Tempo , Transfecção , Adulto Jovem , Quinases da Família src/metabolismo
18.
Cell Rep ; 18(9): 2162-2174, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28249162

RESUMO

BET inhibitors (BETi) target bromodomain-containing proteins and are currently being evaluated as anti-cancer agents. We find that maximal therapeutic effects of BETi in a Myc-driven B cell lymphoma model required an intact host immune system. Genome-wide analysis of the BETi-induced transcriptional response identified the immune checkpoint ligand Cd274 (Pd-l1) as a Myc-independent, BETi target-gene. BETi directly repressed constitutively expressed and interferon-gamma (IFN-γ) induced CD274 expression across different human and mouse tumor cell lines and primary patient samples. Mechanistically, BETi decreased Brd4 occupancy at the Cd274 locus without any change in Myc occupancy, resulting in transcriptional pausing and rapid loss of Cd274 mRNA production. Finally, targeted inhibition of the PD-1/PD-L1 axis by combining anti-PD-1 antibodies and the BETi JQ1 caused synergistic responses in mice bearing Myc-driven lymphomas. Our data uncover an interaction between BETi and the PD-1/PD-L1 immune-checkpoint and provide mechanistic insight into the transcriptional regulation of CD274.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/imunologia , Sistema Imunitário/imunologia , Proteínas Nucleares/imunologia , Receptor de Morte Celular Programada 1/imunologia , Fatores de Transcrição/imunologia , Animais , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Interferon gama/imunologia , Ligantes , Linfoma de Células B/imunologia , Camundongos , Proteínas Proto-Oncogênicas c-myc/imunologia , RNA Mensageiro/imunologia , Transcrição Genética/imunologia
19.
Trends Immunol ; 38(4): 298-305, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28233639

RESUMO

Cancers are often initiated by genetic events that activate proto-oncogenes or inactivate tumor-suppressor genes. These events are also crucial for sustained tumor cell proliferation and survival, a phenomenon described as oncogene addiction. In addition to this cell-intrinsic role, recent evidence indicates that oncogenes also directly regulate immune responses, leading to immunosuppression. Expression of many oncogenes or loss of tumor suppressors induces the expression of immune checkpoints that regulate the immune response, such as PD-L1. We discuss here how oncogenes, and in particular MYC, suppress immune surveillance, and how oncogene-targeted therapies may restore the immune response against tumors.


Assuntos
Carcinogênese , Tolerância Imunológica , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinogênese/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Vigilância Imunológica , Imunomodulação , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Evasão Tumoral
20.
Cytometry B Clin Cytom ; 92(4): 310-314, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-26517296

RESUMO

BACKGROUND: B-lymphoblastic leukemias (B-LBL) with combined IGH/BCL2 and MYC rearrangement are rare and their clinical, cytogenetic and immunophenotypic features are not well characterized. Here, we describe a case of a 61-year-old woman with B-LBL associated with these cytogenetic alterations and present a review of the literature of this disease. METHODS: Four-color flow cytometry (FC) was performed on a BD FACSCanto II flow cytometer. Data were analyzed with BD FACSDiva software. Cytogenetic, fluorescence in situ hybridization (FISH), and molecular studies were performed by conventional methods. A review of the literature was performed by a PubMed-assisted search. RESULTS: Including our case, eight B-LBLs associated with a documented "double-hit" karyotype (IGH/BCL2 and 8q24/MYC rearrangement) were identified in the literature (male/female 2/6, age 15-65). Three occurred de-novo, and five had a history of a CD10+ B-cell lymphoma. The typical immunophenotype was CD10, CD19, TdT positive, and negative for CD34 and surface immunoglobulin (Ig), established either by FC or immunohistochemistry. Seven cases were CD20-, and one case was CD20+. Translocation partners of MYC varied, and included IGH, lambda light chain, and an unknown gene on chromosome 9. Prognosis was poor with median survival of five months. CONCLUSIONS: Patients with B-LBL associated with a combined IGH/BCL2 and MYC rearrangement often have a history of a mature B-cell lymphoma. The immunophenotype of these cases is different from that of mature "double-hit" lymphomas; FC is essential to differentiate the B-LBL cases from the leukemic phase of mature B-cell lymphomas. © 2015 International Clinical Cytometry Society.


Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adolescente , Adulto , Antígenos CD/genética , Antígenos CD/imunologia , Feminino , Citometria de Fluxo/métodos , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Imunofenotipagem , Cariotipagem , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Análise de Sobrevida , Translocação Genética
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