RESUMO
Extra-cellular galectins 1, 3 and 9 (gal-1, -3 and -9) are known to act as soluble immunosuppressive agents in various malignancies. Previous publications have suggested that their expression is dependent on the metabolic status of producing cells and reciprocally that they can influence metabolic pathways in their target cells. Very little is known about the status of gal-1, -3 and -9 in patients bearing head and neck squamous cell carcinomas (HNSCC) and about their relationships with the systemic metabolic condition. This study was conducted in plasma samples from a prospective cohort of 83 HNSCC patients with advanced disease. These samples were used to explore the distribution of gal-1, -3 and -9 and simultaneously to profile a series of 87 metabolites assessed by mass spectrometry. We identified galectin and metabolic patterns within five disease categories defined according to the primary site and human papillomavirus (HPV) status (HPV-positive and -negative oropharyngeal carcinomas, carcinomas of the oral cavity, hypopharynx and larynx carcinomas). Remarkably, samples related to hypopharyngeal carcinomas displayed the highest average concentration of gal-9 (p = .017) and a trend toward higher concentrations of kynurenine, a potential factor of tumor growth and immune suppression. In contrast, there was a tendency toward higher concentrations of fatty acids in samples related to oral cavity. These observations emphasize the diversity of HPV-negative HNSCCs. Depending on their primary site, they evolve into distinct types of immune and metabolic landscapes that seem to be congruent with specific oncogenic mechanisms.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Hipofaríngeas , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Estudos Prospectivos , GalectinasRESUMO
Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones-either positive or negative for gal-9-from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.
Assuntos
Quimiocina CXCL10/genética , Galectinas/genética , Interferon gama/genética , Evasão Tumoral/genética , Neoplasias da Bexiga Urinária/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Nus , Transplante Isogênico , Evasão Tumoral/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.
Assuntos
Aminopiridinas/uso terapêutico , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Triazinas/uso terapêutico , Aminopiridinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Clonais , Estudos de Coortes , Hematopoese , Humanos , Imunofenotipagem , Isocitrato Desidrogenase/metabolismo , Mutação/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Triazinas/farmacologiaRESUMO
Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.
RESUMO
ARHGAP19 is a hematopoietic-specific Rho GTPase-activating protein (RhoGAP) that acts through the RhoA/ROCK pathway to critically regulate cell elongation and cytokinesis during lymphocyte mitosis. We report here that, during mitosis progression, ARHGAP19 is sequentially phosphorylated by the RhoA-activated kinases ROCK1 and ROCK2 (hereafter ROCK) on serine residue 422, and by CDK1 on threonine residues 404 and 476. The phosphorylation of ARHGAP19 by ROCK occurs before mitosis onset and generates a binding site for 14-3-3 family proteins. ARHGAP19 is then phosphorylated by CDK1 in prometaphase. The docking of 14-3-3 proteins to phosphorylated S422 protects ARHGAP19 from dephosphorylation of the threonine sites and prevents ARHGAP19 from relocating to the plasma membrane during prophase and metaphase, thus allowing RhoA to become activated. Disruption of these phosphorylation sites results in premature localization of ARHGAP19 at the cell membrane and in its enrichment to the equatorial cortex in anaphase leading to cytokinesis failure and cell multinucleation.
Assuntos
Citocinese/genética , Proteínas Ativadoras de GTPase/genética , Mitose/genética , Proteína rhoA de Ligação ao GTP/genética , Proteínas 14-3-3/genética , Proteína Quinase CDC2/genética , Humanos , Células Jurkat , Fosforilação/genética , Prometáfase/genética , Serina/genética , Quinases Associadas a rho/genéticaRESUMO
Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including â¼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.
Assuntos
Aminopiridinas/uso terapêutico , Antineoplásicos/uso terapêutico , Glutaratos/metabolismo , Hematopoese/efeitos dos fármacos , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Triazinas/uso terapêutico , Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Feminino , Frequência do Gene , Glutaratos/antagonistas & inibidores , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Triazinas/farmacologiaRESUMO
Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.
Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Triazinas/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Isocitrato Desidrogenase/genética , Camundongos , Mutação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Isocitrate dehydrogenase IDH 1 and IDH 2 mutations were reported in several cancer forms, especially in hematological malignancies, but were never been investigated in familial aggregation. The aim of this study is to determine whether germline isocitrate dehydrogenase genes mutations are involved.We targeted IDH1 and IDH2 genes in 104 familial cases belonging to Tunisian and French populations, including several forms of hematological malignancies and cosegregated solid tumors.We report one IDH1 variant: c.315 G>T, p.Gly105Gly in 15 % of cases, which was assigned to the worst outcome in several studies. Three IDH2 variants were found, among them, one intronic substitution c.543+45 G>A (rs142033117) and two new variants not previously described: c.389 A>T, p.Lys130Met and c.414 T>C, p.Thr138Thr. The p.Lys130Met was found in one case diagnosed with Waldenstrom's disease with familial history of cancer. The enrolled in silico analysis, the functional study, and the absence of this variant in control population strengthen the hypothesis of its deleterious effect.From an extended number of candidate genes analyzed in familial hematological malignancies, IDH2 might be considerably involved since we reported a potential damaging effect.
Assuntos
Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Isocitrato Desidrogenase/genética , Mutação/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.
Assuntos
Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Cardiomiopatias/tratamento farmacológico , Isocitrato Desidrogenase/antagonistas & inibidores , Mutação/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Encefalopatias Metabólicas Congênitas/genética , Modelos Animais de Doenças , Isocitrato Desidrogenase/genética , Camundongos , Mutação/genéticaRESUMO
A recent update of the hallmarks of cancer includes metabolism with deregulating cellular energetics. Activating mutations in isocitrate dehydrogenase (IDH) metabolic enzymes leading to the abnormal accumulation of 2-hydroxyglutaric acid (2-HGA) have been described in hematologic malignancies and solid tumours. The diagnostic value of 2-HGA levels in blood to identify IDH mutations and its prognostic significance have been reported. We developed a liquid chromatography tandem mass spectrometry method allowing a rapid, accurate and precise simultaneous quantification of both L and D enantiomers of 2-HGA in blood samples from acute myeloid leukaemia (AML) patients, suitable for clinical applications. The method was also develop for preclinical applications from cellular and tissues samples. Deuterated (R,S)-2-hydroxyglutaric acid, disodium salt was used as internal standard and added to samples before a solid phase extraction on Phenomenex STRATA™-XL-A (200mg-3mL) 33µm cartridges. A derivatization step with (+)- o,o'-diacetyl-l-tartaric anhydride permitted to separate the two resulting diastereoisomers without chiral stationary phase, on a C18 column combined to a Xevo TQ-MS Waters mass spectrometer with an electrospray ionization (ESI) source. This method allows standard curves to be linear over the range 0.34-135.04µM with r(2) values>0.999 and low matrix effects (<11.7%). This method, which was validated according to current EMA guidelines, is accurate between-run (<3.1%) and within-run (<7.9%) and precise between-run (<5.3CV%) and within-run (<6.2CV%), and is suitable for clinical and preclinical applications.
Assuntos
Biomarcadores Tumorais/sangue , Cromatografia Líquida/métodos , Glutaratos/sangue , Isocitrato Desidrogenase/metabolismo , Espectrometria de Massas em Tandem/métodos , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Glutaratos/química , Glutaratos/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , EstereoisomerismoRESUMO
Suppressor of cytokine signaling (SOCS) 1 is an inducible negative regulator of cytokine signaling but its role in human cancer is not completely established. Here we report that, while SOCS1 is expressed in normal colonic epithelium and colon adenocarcinomas, its level decreases during progression of colon adenocarcinomas, the lowest level being found in the most aggressive stage and least differentiated carcinomas. Forced expression of SOCS1 in metastatic colorectal SW620 cells reverses many characteristics of Epithelial-Mesenchymal Transition (EMT), as highlighted by the disappearance of the transcription factor ZEB1 and the mesenchymal form of p120ctn and the re-expression of E-cadherin. Furthermore, miRNA profiling indicated that SOCS1 also up-regulates the expression of the mir-200 family of miRNAs, which can promote the mesenchymal-epithelial transition and reduce tumor cell migration. Accordingly, overexpression of SOCS1 induced cell morphology changes and dramatically reduced tumor cell invasion in vitro. When injected in nude mice, SOCS1-expressing SW620 cells induced metastases in a smaller number of animals than parental SW620 cells, and did not generate any adrenal gland or bone metastasis. Overall, our results suggest that SOCS1 controls metastatic progression of colorectal tumors by preventing the mesenchymal-epithelial transition (MET), including E-cadherin expression. This pathway may be associated with survival to colorectal cancer by reducing the capacity of generating metastases.
Assuntos
Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Reto/patologia , Proteínas Supressoras da Sinalização de Citocina/genética , Animais , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Reto/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/análise , Proteínas Supressoras da Sinalização de Citocina/metabolismo , TranscriptomaRESUMO
Small GTP-binding proteins of the Rho family orchestrate the cytoskeleton remodelling events required for cell division. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) promote cycling of Rho GTPases between the active GTP-bound and the inactive GDP-bound conformations. We report that ARHGAP19, a previously uncharacterised protein, is predominantly expressed in hematopoietic cells and has an essential role in the division of T lymphocytes. Overexpression of ARHGAP19 in lymphocytes delays cell elongation and cytokinesis. Conversely, silencing of ARHGAP19 or expression of a GAP-deficient mutant induces precocious mitotic cell elongation and cleavage furrow ingression, as well as excessive blebbing. In relation to these phenotypes, we show that ARHGAP19 acts as a GAP for RhoA, and controls recruitment of citron and myosin II to the plasma membrane of mitotic lymphocytes as well as Rock2-mediated phosphorylation of vimentin, which is crucial to maintain the stiffness and shape of lymphocytes. In addition to its effects on cell shape, silencing of ARHGAP19 in lymphocytes also impairs chromosome segregation.
Assuntos
Segregação de Cromossomos , Citocinese , Proteínas Ativadoras de GTPase/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Forma Celular/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia/genética , Leucemia/patologia , Mitose/efeitos dos fármacos , Mitose/genética , Miosina Tipo II/metabolismo , Nocodazol/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Prometáfase/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Vimentina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The dynamics of the actin cytoskeleton and its regulation by Rho GTPases are essential to maintain cell shape, to allow cell motility and are also critical during cell cycle progression and mitosis. Rho GTPases and their effectors are involved in cell rounding at mitosis onset, in chromosomes alignment and are required for contraction of the actomyosin ring that separates daughter cells at the end of mitosis. Recent studies have revealed how a number of nucleotide exchange factors and GTPase-activating proteins regulate the activity of Rho GTPases during these processes. This review will focus on how the cell cycle machinery, in turn, regulates expression of proteins in the Rho signaling pathways through transcriptional activation, ubiquitylation and proteasomal degradation and modulates their activity through phosphorylation by mitotic kinases.
Assuntos
Ciclo Celular , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Diferenciação Celular , Ativação Enzimática , Humanos , Megacariócitos/citologia , Megacariócitos/enzimologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fuso Acromático/genética , Fuso Acromático/metabolismo , Ativação Transcricional , Ubiquitinação , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Histone deacetylase inhibitors (HDACi) have been hailed as a powerful new class of anticancer drugs. The HDACi, trichostatin A (TSA), is thought to interfere with epigenetic control of cell cycle progression in G1 and G2-M phase, resulting in growth arrest, differentiation, or apoptosis. Here, we describe a novel mechanism of action of HDACis in promoting immune responses against tumors. We report that treatment of carcinoma cells with TSA increases the expression of many components of the antigen processing machinery, including TAP-1, TAP-2, LMP-2, and Tapasin. Consistent with this result, we found that treatment of metastatic carcinoma cells with TSA also results in an increase in MHC class I expression on the cell surface that functionally translates into an enhanced susceptibility to killing by antigen-specific CTLs. Finally, we observed that TSA treatment suppresses tumor growth and increases tap-1 promoter activity in TAP-deficient tumor cells in vivo. Intriguingly, this in vivo anti-tumoral effect of TSA is entirely mediated by an increase in immunogenicity of the tumor cells, as it does not occur in immunodeficient mice. These novel insights into the molecular mechanisms controlling tumor immune escape may help revise immunotherapeutic modalities for eradicating cancers.
Assuntos
Antígenos de Neoplasias/imunologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Antígenos de Neoplasias/biossíntese , Linhagem Celular Tumoral , Epigênese Genética/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Melanoma Experimental/genética , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Downregulation of the transporter associated with antigen processing 1 (TAP-1) has been observed in many tumors and is closely associated with tumor immunoevasion mechanisms, growth, and metastatic ability. The molecular mechanisms underlying the relatively low level of transcription of the tap-1 gene in cancer cells are largely unexplained. In this study, we tested the hypothesis that epigenetic regulation plays a fundamental role in controlling tumor antigen processing and immune escape mechanisms. We found that the lack of TAP-1 transcription in TAP-deficient cells correlated with low levels of recruitment of the histone acetyltransferase, CBP, to the TAP-1 promoter. This results in lower levels of histone H3 acetylation at the TAP-1 promoter, leading to a decrease in accessibility of the RNA polymerase II complex to the TAP-1 promoter. These observations suggest that CBP-mediated histone H3 acetylation normally relaxes the chromatin structure around the TAP-1 promoter region, allowing transcription. In addition, we found a hitherto-unknown mechanism wherein interferon gamma up-regulates TAP-1 expression by increasing histone H3 acetylation at the TAP-1 promoter locus. These findings lie at the heart of understanding immune escape mechanisms in tumors and suggest that the reversal of epigenetic codes may provide novel immunotherapeutic paradigms for intervention in cancer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica , Metástase Neoplásica , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Acetilação , Animais , Sequência de Bases , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Carcinoma/genética , Carcinoma/imunologia , Carcinoma/patologia , Linhagem Celular , Genes MHC Classe I , Histonas/metabolismo , Humanos , Interferon gama/metabolismo , Complexo Principal de Histocompatibilidade , Camundongos , Dados de Sequência Molecular , Invasividade Neoplásica/genética , Invasividade Neoplásica/imunologia , Metástase Neoplásica/genética , Metástase Neoplásica/imunologia , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G(1)-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF-2alpha) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Movimento Celular , Sequência Conservada , Ciclina D , Ciclinas/genética , Grânulos Citoplasmáticos/efeitos dos fármacos , DNA Helicases , Regulação da Expressão Gênica , Humanos , Camundongos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismo , Alinhamento de SequênciaRESUMO
Recognition of bacterial LPS by macrophages plays a critical role in host defense against infection by Gram-negative bacteria. However, when not tightly regulated, the macrophage's response to LPS can induce severe disease and septic shock. Although LPS triggers the activation of multiple signaling pathways in macrophages, it was unclear whether these include activation of the p21Ras GTPases. We report that p21Ras is rapidly and transiently activated in murine primary macrophages stimulated with an ultra-pure preparation of LPS or with synthetic lipid A. The molecular basis of this activation was investigated using a pharmacological approach. LPS-induced activation of p21Ras was inhibited in the presence of PP2, LY294002, or wortmannin, suggesting that it depends on the activity of one or more members of the Src kinase family and the subsequent activation of PI3K. In that pharmacological inhibitors of PI3K inhibited LPS-induced activation of p21Ras, but not activation of ERK, we concluded that LPS-induced activation of ERK occurs through a pathway that is not dependent on the activation of p21Ras.
Assuntos
Lipídeo A/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células da Medula Óssea , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Quinases da Família src/metabolismoRESUMO
Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is a cytoplasmic phosphoprotein that is the prototype of a novel family of highly conserved proteins. Its levels, except in the brain, are tightly correlated with cellular proliferation. We disrupted caprin-1 alleles in the chicken B lymphocyte line DT40 using homologous recombination. We readily obtained clones with one disrupted allele (31% of transfectants), but upon transfection of heterozygous cells we obtained a 10-fold lower frequency of clones with disruption of the remaining allele. Clones of caprin-1-null DT40 cells exhibited marked reductions in their proliferation rate. To obviate the problem that we had selected for caprin-1-null clones with characteristics that partially compensated for the lack of Caprin-1, we generated clones of DT40 cells heterozygous for the caprin-1 gene in which, during disruption of the remaining wild-type allele of the chicken caprin-1 gene, the absence of endogenous Caprin-1 would be complemented by conditional expression of human Caprin-1. Suppression of expression of human Caprin-1 resulted in slowing of the proliferation rate, due to prolongation of the G1 phase of the cell cycle, formally demonstrating that Caprin-1 was essential for normal cellular proliferation.
Assuntos
Proteínas de Ciclo Celular/genética , Proliferação de Células , Fosfoproteínas/genética , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular , Galinhas , Sequência Conservada , DNA Complementar/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fase G1/genética , Marcação de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/deficiência , Fosforilação , Alinhamento de Sequência , TransfecçãoRESUMO
Expression of transporter associated with antigen processing (TAP) is often lost in metastatic carcinomas, resulting in defective antigen processing and presentation and escape of the cancer cells from immune surveillance. In this study, the nature of TAP deficiencies in tumors was investigated. By chromatin immunoprecipitation assay, we showed that the recruitment of RNA polymerase II to the TAP-1 gene was impaired in TAP-deficient cells derived from murine melanoma, prostate, and lung carcinomas, compared with TAP-expressing fibroblasts and lymphoma cells. This suggested that the deficiency in TAP-1 expression resulted, at least partially, from a relatively low level of transcription of the TAP-1 gene. Furthermore, levels of TAP-1 promoter activity, as assessed by stable transfections with a reporter construct containing the TAP-1 promoter, were relatively low in TAP-deficient cells. To examine genetic heritability of regulators of TAP-1 promoter activity, TAP- and MHC class I-deficient cells of H-2b origin were fused with wild-type fibroblasts of H-2k origin. Fusion with TAP-expressing cells complemented the low levels of TAP-1 promoter activity in TAP-deficient cells. However, these fused cells exhibited lower levels of TAP-1 mRNA and H-2k than unfused fibroblasts. Further analysis showed that TAP-1 mRNA stability was lower in fused carcinoma fibroblasts than in unfused fibroblasts. Based on these results, we propose that TAP deficiency in many carcinomas is caused by a decrease in activity/expression of trans-acting factors regulating TAP-1 promoter activity, as well as a decrease in TAP-1 mRNA stability. These results have significant implications for understanding immune evasion mechanisms in tumors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos de Neoplasias/imunologia , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Neoplasias Experimentais/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Sequência de Bases , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Linfoma/genética , Linfoma/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Neoplasias Experimentais/genética , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Transcrição Gênica , TransfecçãoRESUMO
Although GTPases of the Ras family have been implicated in many aspects of the regulation of cells, little is known about the roles of individual family members. Here, we analyzed the mechanisms of activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by two types of external stimuli, growth factors and ligation of the antigen receptors of B or T lymphocytes (BCRs and TCRs). The growth factors interleukin-3, colony-stimulating factor 1, and epidermal growth factor all preferentially activated M-Ras and K-Ras 4B over H-Ras or N-Ras. Preferential activation of M-Ras and K-Ras 4B depended on the presence of their polybasic carboxy termini, which directed them into high-buoyant-density membrane domains where the activated receptors, adapters, and mSos were also present. In contrast, ligation of the BCR or TCR resulted in activation of H-Ras, N-Ras, and K-Ras 4B, but not M-Ras. This pattern of activation was not influenced by localization of the Ras proteins to membrane domains. Activation of H-Ras, N-Ras, and K-Ras 4B instead depended on the presence of phospholipase C-gamma and RasGRP. Thus, the molecular mechanisms leading to activation of Ras proteins vary with the stimulus and can be influenced by either colocalization with activated receptors or differential sensitivity to the exchange factors activated by a stimulus.