RESUMO
Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.
Assuntos
Doenças Autoimunes/imunologia , Disbiose/imunologia , Inflamação/imunologia , Interferon gama/metabolismo , Macrófagos/imunologia , Regiões 3' não Traduzidas/genética , Elementos Ricos em Adenilato e Uridilato/genética , Animais , Autofagia , Doença Crônica , Feminino , Microbioma Gastrointestinal/imunologia , Interferon gama/genética , Masculino , Camundongos , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/metabolismo , Sexismo , Transdução de SinaisRESUMO
During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.
Assuntos
Linfócitos B/citologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/imunologia , Citidina Desaminase/genética , Dano ao DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos da radiação , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/efeitos da radiação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica/imunologia , Centro Germinativo/citologia , Humanos , Switching de Imunoglobulina/fisiologia , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Plasmócitos/citologia , Plasmócitos/imunologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) causes progressive loss of renal function in adults as a consequence of the accumulation of cysts. ADPKD is the most common genetic cause of end-stage renal disease. Mutations in polycystin-1 occur in 87% of cases of ADPKD and mutations in polycystin-2 are found in 12% of ADPKD patients. The complexity of ADPKD has hampered efforts to identify the mechanisms underlying its pathogenesis. No current FDA (Federal Drug Administration)-approved therapies ameliorate ADPKD progression. RESULTS: We used the de Almeida laboratory's sensitive new transcriptogram method for whole-genome gene expression data analysis to analyze microarray data from cell lines developed from cell isolates of normal kidney and of both non-cystic nephrons and cysts from the kidney of a patient with ADPKD. We compared results obtained using standard Ingenuity Volcano plot analysis, Gene Set Enrichment Analysis (GSEA) and transcriptogram analysis. Transcriptogram analysis confirmed the findings of Ingenuity, GSEA, and published analysis of ADPKD kidney data and also identified multiple new expression changes in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways related to cell growth, cell death, genetic information processing, nucleotide metabolism, signal transduction, immune response, response to stimulus, cellular processes, ion homeostasis and transport and cofactors, vitamins, amino acids, energy, carbohydrates, drugs, lipids, and glycans. Transcriptogram analysis also provides significance metrics which allow us to prioritize further study of these pathways. CONCLUSIONS: Transcriptogram analysis identifies novel pathways altered in ADPKD, providing new avenues to identify both ADPKD's mechanisms of pathogenesis and pharmaceutical targets to ameliorate the progression of the disease.
Assuntos
Rim Policístico Autossômico Dominante/metabolismo , Transcriptoma , Adulto , Estudos de Casos e Controles , Linhagem Celular , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of ß-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.
Assuntos
Proteínas de Transporte/metabolismo , Transplante de Rim , Rim/metabolismo , Insuficiência Renal/prevenção & controle , Proteínas Wnt/metabolismo , Actinas/metabolismo , Animais , Anticorpos/uso terapêutico , Biomarcadores/urina , Caderinas/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Rim/imunologia , Testes de Função Renal , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Insuficiência Renal/urina , beta Catenina/metabolismoRESUMO
Drug resistance is a long-standing impediment to effective systemic cancer therapy and acquired drug resistance is a growing problem for molecularly-targeted therapeutics that otherwise have shown unprecedented successes in disease control. The hepatocyte growth factor (HGF)/Met receptor pathway signaling is frequently involved in cancer and has been a subject of targeted drug development for nearly 30 years. To anticipate and study specific resistance mechanisms associated with targeting this pathway, we engineered resistance to the HGF-neutralizing antibody rilotumumab in glioblastoma cells harboring autocrine HGF/Met signaling, a frequent abnormality of this brain cancer in humans. We found that rilotumumab resistance was acquired through an unusual mechanism comprising dramatic HGF overproduction and misfolding, endoplasmic reticulum (ER) stress-response signaling and redirected vesicular trafficking that effectively sequestered rilotumumab and misfolded HGF from native HGF and activated Met. Amplification of MET and HGF genes, with evidence of rapidly acquired intron-less, reverse-transcribed copies in DNA, was also observed. These changes enabled persistent Met pathway activation and improved cell survival under stress conditions. Point mutations in the HGF pathway or other complementary or downstream growth regulatory cascades that are frequently associated with targeted drug resistance in other prevalent cancer types were not observed. Although resistant cells were significantly more malignant, they retained sensitivity to Met kinase inhibition and acquired sensitivity to inhibition of ER stress signaling and cholesterol biosynthesis. Defining this mechanism reveals details of a rapidly acquired yet highly-orchestrated multisystem route of resistance to a selective molecularly-targeted agent and suggests strategies for early detection and effective intervention.
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Multiple myeloma (MM) is a plasma cell malignancy expressing B cell maturation antigen (BCMA). Elranatamab, a bispecific antibody, engages BCMA on MM and CD3 on T cells. The MagnetisMM-1 trial evaluated its safety, pharmacokinetics and efficacy. Primary endpoints, including the incidence of dose-limiting toxicities as well as objective response rate (ORR) and duration of response (DOR), were met. Secondary efficacy endpoints included progression-free survival (PFS) and overall survival (OS). Eighty-eight patients with relapsed or refractory MM received elranatamab monotherapy, and 55 patients received elranatamab at efficacious doses. Patients had received a median of five prior regimens; 90.9% were triple-class refractory, 29.1% had high cytogenetic risk and 23.6% received prior BCMA-directed therapy. No dose-limiting toxicities were observed during dose escalation. Adverse events included cytopenias and cytokine release syndrome. Exposure was dose proportional. With a median follow-up of 12.0 months, the ORR was 63.6% and 38.2% of patients achieving complete response or better. For responders, the median DOR was 17.1 months. All 13 patients evaluable for minimal residual disease achieved negativity. Even after prior BCMA-directed therapy, 53.8% achieved response. For all 55 patients, median PFS was 11.8 months, and median OS was 21.2 months. Elranatamab achieved durable responses, manageable safety and promising survival for patients with MM. ClinicalTrials.gov Identifier: NCT03269136 .
Assuntos
Anemia , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/patologia , Antígeno de Maturação de Linfócitos B , Linfócitos T/patologia , Intervalo Livre de Progressão , Anemia/etiologia , Imunoterapia Adotiva/efeitos adversosRESUMO
PURPOSE: Epidermal growth factor receptor variant III (EGFRvIII) is expressed in a significant percentage of primary and recurrent glioblastoma (GBM), a common malignant primary brain tumor in adults. AMG 595 is an antibody-drug conjugate comprising a fully human, anti-EGFRvIII monoclonal antibody linked to DM1. The study goals were to assess safety, tolerability, and pharmacokinetics of AMG 595 in GBM. METHODS: In this phase 1, first-in-human, open-label, sequential-dose, exploration study, adults with recurrent GBM received AMG 595 once every 3 weeks (Q3W) according to incremental dosing cohorts (0.5-3.0 mg/kg). Primary endpoints were to assess safety, the incidence of dose-limiting toxicities (DLTs), objective response (per Macdonald criteria), evaluate pharmacokinetics, and estimate the maximum tolerated dose (MTD). RESULTS: Of 382 patients screened, 32 were enrolled and received ≥ 1 dose of AMG 595. Ten patients experienced 18 DLTs (all grade 4 thrombocytopenia), and the MTD was 2.0 mg/kg. Twenty-eight patients (88%) experienced ≥ 1 treatment-related adverse event (AE); the most common AEs were thrombocytopenia (50%) and fatigue (25%). Grade ≥ 3 treatment-related AEs occurred in 17 patients (53%); 11 (34%) had serious treatment-emergent AEs, and none were considered treatment related. Pharmacokinetic profiles indicated low levels of circulating unconjugated antibody and cytotoxin, dose-proportional increases in plasma exposures for the conjugated antibody over the studied range, and less than twofold accumulation following multiple Q3W dosing. Two patients (6%) had partial responses; 15 (47%) had stable disease. CONCLUSIONS: AMG 595 exhibited favorable pharmacokinetics and is a unique therapy with possible benefit for some patients with EGFRvIII-mutated GBM with limited therapeutic options.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Imunoconjugados/uso terapêutico , Maitansina/análogos & derivados , Adulto , Idoso , Neoplasias Encefálicas/patologia , Feminino , Glioma/patologia , Humanos , Imunoconjugados/farmacocinética , Masculino , Maitansina/farmacocinética , Maitansina/uso terapêutico , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.
Assuntos
Perfilação da Expressão Gênica/normas , Análise de Sequência com Séries de Oligonucleotídeos/normas , RNA Mensageiro/normas , Animais , Perfilação da Expressão Gênica/métodos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
OBJECTIVE: Interferon-γ (IFNγ) is implicated in the pathogenesis of discoid lupus erythematosus (DLE). This study sought to evaluate a single dose of AMG 811, an anti-IFNγ antibody, in patients with DLE. METHODS: The study was designed as a phase I randomized, double-blind, placebo-controlled crossover study of the pharmacodynamics, safety, and clinical efficacy of AMG 811 in patients with DLE. Patients received a single subcutaneous dose of AMG 811 (180 mg) or placebo. The patients in sequence 1 received AMG 811 followed by placebo, while those in sequence 2 received placebo followed by AMG 811. Pharmacodynamic end points included global transcriptional analyses of lesional and nonlesional skin, IFNγ blockade signature (IGBS) transcriptional scores in the skin and blood, keratinocyte IFNγ RNA scores, and serum levels of CXCL10 protein. Additional end points were efficacy outcome measures, including the Cutaneous Lupus Erythematosus Disease Area and Severity Index, and safety outcome measures. RESULTS: Sixteen patients with DLE were enrolled in the study (9 in sequence 1 and 7 in sequence 2). AMG 811 treatment reduced the IGBS score (which was elevated in DLE patients at baseline) in both the blood and lesional skin. The keratinocyte IFNγ RNA score was not affected by administration of AMG 811. Serum CXCL10 protein levels (which were elevated in the blood of DLE patients) were reduced with AMG 811 treatment. The AMG 811 treatment was well tolerated but did not lead to statistically significant improvements in any of the efficacy outcome measures. CONCLUSION: AMG 811 treatment led to changes in IFNγ-associated biomarkers and was well tolerated, but no significant clinical benefit was observed in patients with DLE.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunossupressores/uso terapêutico , Interferon gama/imunologia , Lúpus Eritematoso Discoide/tratamento farmacológico , Adulto , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Quimiocina CXCL10/imunologia , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Imunossupressores/farmacocinética , Interferon gama/genética , Lúpus Eritematoso Discoide/imunologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Resultado do TratamentoRESUMO
OBJECTIVE: To evaluate safety, pharmacokinetics and pharmacodynamics of anti-interferon (IFN)-γ monoclonal antibody AMG 811 in subjects with SLE without or with lupus nephritis (LN). METHODS: In this phase Ib, randomised, multiple-dose escalation study (NCT00818948), subjects without LN were randomised to subcutaneous AMG 811 (6, 20 or 60 mg) or placebo and subjects with LN were randomised to subcutaneous AMG 811 (20, 60 or 120 mg) or placebo every four weeks for three total doses. Outcomes included incidence of adverse events (AEs); pharmacokinetics; levels of serum proteins (CXCL-10, interleukin 18, monocyte chemotactic protein-1); changes in gene transcript profiles and clinical parameters (Safety of Estrogen in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) scores, proteinuria, anti-double-stranded DNA (anti-dsDNA) antibodies, C3 complement, C4 complement). RESULTS: Fifty-six subjects enrolled (28 SLE without LN; 28 with LN). Baseline mean SELENA-SLEDAI scores were 2.2 and 12.0 for SLE subjects without and with LN, respectively. Most subjects reported an AE; no meaningful imbalances were observed between AMG 811 and placebo. Pharmacokinetic profiles were similar and mostly dose-proportional in subjects without or with LN. AMG 811 treatment reduced CXCL-10 protein levels and blood-based RNA IFN-γ Blockade Signature compared with placebo. Reductions were less pronounced and not sustained in subjects with LN, even at the highest dose tested, compared with subjects without LN. No effect on SELENA-SLEDAI scores, proteinuria, C3 or C4 complement levels, or anti-dsDNA antibodies was observed. CONCLUSION: AMG 811 demonstrated favourable pharmacokinetics and acceptable safety profile but no evidence of clinical impact. IFN-γ-associated biomarkers decreased with AMG 811; effects were less pronounced and not sustained in LN subjects. TRIAL REGISTRATION NUMBER: NCT00818948; results.
RESUMO
The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568-79. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Amplificação de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Necrose , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-beta) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-beta1 on breast cancer cells in the presence or absence of overexpressed HER-2. METHODS: Cell proliferation assays were used to determine the effect of TGF-beta on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-beta1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-beta signaling pathway was assessed using TGF-beta1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. RESULTS: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-beta1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-beta in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-beta induced pro-invasive and pro-metastatic gene signature. CONCLUSION: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-beta. In contrast, HER-2 and TGF-beta signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.
Assuntos
Neoplasias da Mama/genética , Receptor ErbB-2/biossíntese , Fator de Crescimento Transformador beta/fisiologia , Northern Blotting , Western Blotting , Neoplasias da Mama/patologia , Proliferação de Células , Progressão da Doença , Células Epiteliais , Feminino , Perfilação da Expressão Gênica , Engenharia Genética , Humanos , Glândulas Mamárias Humanas/citologia , Mesoderma , Invasividade Neoplásica , Transdução de Sinais , Fator de Crescimento Transformador beta1RESUMO
Interferon-gamma (IFN gamma) exerts diverse responses in B cell development ranging from growth arrest and apoptosis to proliferation and differentiation. IFN gamma stimulates murine 70Z/3 pre-B cells to express surface immunoglobulin (Ig) and this system serves as a useful model for the pre-B to immature B cell transition in B cell development. To analyze this developmental transition, we used a PCR-based subtractive hybridization in combination with miniarray screening to identify differentially-expressed genes in IFN gamma-stimulated compared with unstimulated 70Z/3 pre-B cells. The majority (44%) of the differentially-expressed genes obtained were known IFN gamma-inducible. These included multiple isolates from each of three multi-gene families, including two guanylate-binding protein (47 and 67kDa GBP) families of GTPases and the hematopoietic IFN gamma-inducible nuclear protein family (HIN-200). These multiple isolates of genes comprised the majority of the total isolated and sequenced clones. Other known IFN gamma-induced genes in this group included Ig kappa light chain and Ly-6, as well as genes with functions in antigen processing, cellular regulation, and cytoskeletal organization. Another 36% of the genes identified were previously known, but not known to be IFN gamma-inducible (e.g. pre-B cell enhancing factor, PBEF). The remaining 20% of the IFN gamma-induced isolates did not match entries in Genbank, and thus, may represent novel genes involved in IFN gamma responses and/or in the pre-B to immature B cell transition. Overall, the majority of the individual genes isolated were either not known to be IFN gamma responsive or were not previously known.
Assuntos
Linfócitos B/imunologia , Interferon gama/farmacologia , Ativação Transcricional , Animais , Linfócitos B/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Células-Tronco/efeitos dos fármacosRESUMO
OBJECTIVE: To assess the safety and immunologic impact of inhibiting interferon-γ (IFNγ) with AMG 811, a human IgG1 monoclonal antibody against IFNγ, in patients with systemic lupus erythematosus (SLE). METHODS: Twenty-six patients with mild-to-moderate, stable SLE were administered placebo or a single dose of AMG 811, ranging from 2 mg to 180 mg subcutaneously or 60 mg intravenously. RESULTS: Similar to results previously reported following inhibition of type I IFNs, treatment of SLE patients with AMG 811 led to a dose-dependent modulation of the expression of genes associated with IFN signaling, as assessed by microarray analysis of the whole blood. The list of impacted genes overlapped with that identified by stimulating human whole blood with IFNγ and with those gene sets reported in the literature to be differentially expressed in SLE patients. Serum levels of IFNγ-induced chemokines, including IFNγ-inducible protein 10 (IP-10), were found to be elevated at baseline in SLE patients as compared to healthy volunteers. In contrast to previously reported results from studies using type I IFN-blocking agents, treatment with AMG 811 led to dose-related reductions in the serum levels of CXCL10 (IP-10). CONCLUSION: The scope and nature of the biomarkers impacted by AMG 811 support targeting of IFNγ as a therapeutic strategy for SLE.
Assuntos
Anticorpos Monoclonais/farmacologia , Quimiocina CXCL10/sangue , Quimiocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/antagonistas & inibidores , Interferons/fisiologia , Lúpus Eritematoso Sistêmico/metabolismo , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Quimiocina CXCL10/genética , Quimiocina CXCL10/fisiologia , Quimiocinas/fisiologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Interferon gama/efeitos dos fármacos , Interferon gama/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resultado do TratamentoRESUMO
We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corrosion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.
Assuntos
Cromossomos Humanos/genética , Lipossarcoma/genética , Neoplasias Retroperitoneais/genética , Idoso , Carcinogênese/genética , Linhagem Celular Tumoral , Centrômero/genética , Aberrações Cromossômicas , Feminino , Humanos , Lipossarcoma/patologia , Modelos Genéticos , Mutagênese , Oncogenes , Neoplasias Retroperitoneais/patologia , Translocação GenéticaRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR)-targeted agents have demonstrated clinical benefit in patients with cancer. Identifying tissue-of-origin-independent predictive biomarkers is important to optimally treat patients. We sought to identify a gene array profile that could predict responsiveness to panitumumab, a fully human EGFR-binding antibody, using preclinical models of human cancer. METHODS: Mice bearing 25 different xenograft models were treated twice weekly with panitumumab or immunoglobulin G2 control to determine their responsiveness to panitumumab. Samples from these xenografts and untreated xenografts were arrayed on the Affymetrix human U133A gene chip to identify gene sets predicting responsiveness to panitumumab using univariate and multivariate analyses. The predictive models were validated using the leave-one-group-out (LOO) method. RESULTS: Of the 25 xenograft models tested, 12 were responsive and 13 were resistant to panitumumab. Unsupervised analysis demonstrated that the xenograft models clustered by tissue type rather than responsiveness to panitumumab. After normalizing for tissue effects, samples clustered by responsiveness using an unsupervised multidimensional scaling. A multivariate selection algorithm was used to select 13 genes that could stratify xenograft models based on responsiveness after adjustment for tissue effects. The method was validated using the LOO method on a training set of 22 models and confirmed independently on three new models. In contrast, a univariate gene selection method resulted in higher misclassification rates. CONCLUSION: A model was constructed from microarray data that prospectively predict responsiveness to panitumumab in xenograft models. This approach may help identify patients, independent of disease origin, likely to benefit from panitumumab.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Biomarcadores Farmacológicos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/patologia , Panitumumabe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sclerostin is a secreted inhibitor of Wnt signaling and plays an essential role in the regulation of bone mass. The expression of sclerostin is largely restricted to osteocytes although its mode of transcriptional regulation is not well understood. We observed regulated expression of sclerostin mRNA and protein that was directly correlated with the mineralization response in cultured human Saos-2 osteosarcoma cells and rat primary calvarial cells. Sclerostin mRNA and protein levels were increased following treatment of cells with BMP2, BMP4 and BMP7. Analysis of deletion mutants from the -7.4 kb upstream region of the human sclerostin promoter did not reveal any specific regions that were responsive to BMPs, Wnt3a, PTH, TGFß1 or Activin A in Saos-2 cells. The downstream ECR5 element did not show enhancer activity in Saos-2 cells and also was not affected when Saos-2 cells were treated with BMPs or PTH. Genome-wide microarray analysis of Saos-2 cells treated with BMP2 showed significant changes in expression of several transcription factors with putative consensus DNA binding sites in the region of the sclerostin promoter. However, whereas most factors tested showed either a range of inhibitory activity (DLX family, MSX2, HEY1, SMAD6/7) or lack of activity on the sclerostin promoter including SMAD9, only MEF2B showed a positive effect on both the promoter and ECR5 element. These results suggest that the dramatic induction of sclerostin gene expression by BMPs in Saos-2 cells occurs indirectly and is associated with late stage differentiation of osteoblasts and the mineralization process.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Elementos Facilitadores Genéticos/genética , Marcadores Genéticos/genética , Osteossarcoma/genética , Regiões Promotoras Genéticas/genética , Ativinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteossarcoma/patologia , Hormônio Paratireóideo/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteína Wnt3A/farmacologiaRESUMO
BACKGROUND: C57BLKS/J (BLKS) mice are susceptible to islet exhaustion in insulin-resistant states as compared with C57BL6/J (B6) mice, as observed by the presence of the leptin receptor (Lepr) allele, Leprdb/db. Furthermore, DBA2/J (DBA) mice are also susceptible to beta-cell failure and share 25% of their genome with BLKS; thus the DBA genome may contribute to beta-cell dysfunction in BLKS mice. RESULTS: Here we show that BLKS mice exhibit elevated insulin secretion, as evidenced by improved glucose tolerance and increased islet insulin secretion compared with B6 mice, and describe interstrain transcriptional differences in glucose response. Transcriptional differences between BLKS and B6 mice were identified by expression profiling of isolated islets from both strains. Genomic mapping of gene expression differences demonstrated a significant association of expression differences with DBA loci in BLKS mice (P = 4x10-27). CONCLUSION: Two genes, Nicotinamide nucleotide transhydrogenase (Nnt) and Pleiomorphic adenoma gene like 1 (Plagl1), were 4 and 7.2-fold higher respectively in BLKS islets, and may be major contributors to increased insulin secretion by BLKS islets. Contrary to reports for B6 mice, BLKS mice do not harbor a mutant Nnt gene. We detected 16 synonymous polymorphisms and a two-amino acid deletion in the Plagl1 gene in BLKS mice. Several inflammatory glucose-responsive genes are expressed at a higher level in BLKS, suggesting an inflammatory component to BLKS islet dysfunction. This study describes physiological differences between BLKS and B6 mice, and provides evidence for a causative role of the DBA genome in beta-cell dysfunction in BLKS mice.
RESUMO
We conducted a genome-wide screen using the mutagen N-ethyl-N-nitrosourea to identify recessive mutations in genes that lead to altered lipid traits in mice. We screened 7,546 G3 mice that were of mixed C57BL/6J (B6) x C3.SW-H2(b)/SnJ (C3) genomes and identified three pedigrees with differences in plasma HDL-cholesterol. Genome scan analyses mapped three distinct loci to chromosomes 3, 4, and 7. An S1748L missense mutation was identified in ABCA1 in one pedigree with undetectable levels of HDL-cholesterol and resulted in reduced protein levels. This phenotype was completely penetrant, semi-dominant, and cosegregated with high plasma triglycerides. Mice in a second pedigree had very high levels of plasma total cholesterol and HDL-cholesterol (up to 800 mg/dl total cholesterol). Despite a high degree of phenotype lability and reduced penetrance, an I68N missense mutation was identified in the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). Finally, a second high HDL-cholesterol pedigree of mice, again with a highly labile phenotype and reduced penetrance, was mapped to a 7 Mb locus on chromosome 3. These results illustrate the use of a hybrid background for simultaneous screening and mapping of mutagenized pedigrees of mice and identification of three novel alleles of HDL-cholesterol phenotypes.
Assuntos
HDL-Colesterol/sangue , HDL-Colesterol/genética , Genes Recessivos , Mutação , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Colesterol/deficiência , Mapeamento Cromossômico , DNA/genética , Etilnitrosoureia/toxicidade , Feminino , Testes Genéticos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese , Mutagênicos/toxicidade , Mutação de Sentido Incorreto , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The formation of Alzheimer's Abeta peptide is initiated when the amyloid precursor protein (APP) is cleaved by the enzyme beta-secretase (BACE1); inhibition of this cleavage has been proposed as a means of treating Alzheimer's disease. (AD) We have previously shown that young BACE1 knockout mice (BACE1 KO) do not generate Abeta but in other respects appear normal. Here we have extended this analysis to include both gene expression profiling and phenotypic assessment of older BACE1 KO animals to evaluate the impact of chronic Abeta deficiency. We did not detect global compensatory changes in neural gene expression in young BACE1 KO mice. In particular, expression of the beta-secretase homolog BACE2 was not upregulated. Furthermore, we found no structural alterations in any organ, including all central and peripheral neural tissues, of BACE1 KO mice up to 14 months of age. Aged BACE1 KO mice engineered to overexpress human APP (BACE1 KO/APPtg) did not develop amyloid plaques. These data provide evidence that neither beta-secretase nor Abeta plays a vital role in mouse physiology and that chronic beta-secretase inhibition could be a useful approach in treating AD.