RESUMO
Reactive oxygen species (ROS) are central effectors of inflammation and play a key role in cell signaling. Previous reports have described an association between oxidative events and the modulation of innate immunity. However, the role of redox signaling in adaptive immunity is still not well understood. This work is based on a novel investigation of diamide, a specific oxidant of sulfhydryl groups, and it is the first performed in purified T cell tyrosine phosphorylation signaling. Our data show that ex vivo T cells respond to -SH group oxidation with a distinctive tyrosine phosphorylation response and that these events elicit specific cellular responses. The expression of two essential T-cell receptors, CD25 and CD62L, and T-cell cytokine release is also affected in a specific way. Experiments with Syk inhibitors indicate a major contribution of this kinase in these phenomena. This pilot work confirms the presence of crosstalk between oxidation of cysteine residues and tyrosine phosphorylation changes, resulting in a series of functional events in freshly isolated T cells. Our experiments show a novel role of Syk inhibitors in applying their anti-inflammatory action through the inhibition of a ROS-generated reaction.
Assuntos
Selectina L/metabolismo , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/fisiologia , Quinase Syk/metabolismo , Linfócitos T , Sobrevivência Celular , Células Cultivadas , Diamida , Humanos , Oxirredução , Fosforilação , Linfócitos T/metabolismo , Linfócitos T/fisiologiaRESUMO
BACKGROUND: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression. CONCLUSIONS: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.
Assuntos
Antígeno B7-H1/efeitos dos fármacos , Interferon gama/farmacologia , Neoplasias/genética , Proteína 2 Ligante de Morte Celular Programada 1/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Evasão Tumoral/efeitos dos fármacos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Janus Quinases/efeitos dos fármacos , Janus Quinases/metabolismo , Neoplasias Hepáticas/secundário , Terapia de Alvo Molecular , Neoplasias/metabolismo , Organoides , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Interferon , Fator de Transcrição STAT1/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Evasão Tumoral/genética , Receptor de Interferon gamaRESUMO
Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.
Assuntos
Astrócitos/fisiologia , Reconhecimento Psicológico/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Ondas Encefálicas/efeitos dos fármacos , Ondas Encefálicas/fisiologia , Sinalização do Cálcio , Carbacol/farmacologia , Eletroencefalografia , Expressão Gênica , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmissão Sináptica , Toxina Tetânica/genética , Toxina Tetânica/metabolismo , Técnicas de Cultura de Tecidos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
RARA becomes an acute promyelocytic leukemia (APL) oncogene by fusion with any of five translocation partners. Unlike RARalpha, the fusion proteins homodimerize, which may be central to oncogenic activation. This model was tested by replacing PML with dimerization domains from p50NFkappaB (p50-RARalpha) or the rapamycin-sensitive dimerizing peptide of FKBP12 (F3-RARalpha). The X-RARalpha fusions recapitulated in vitro activities of PML-RARalpha. For F3-RARalpha, these properties were rapamycin sensitive. Although in vivo the artificial fusions alone are poor initiators of leukemia, p50-RARalpha readily cooperates with an activated mutant CDw131 to induce APL-like disease. These results demonstrate that the dimerization interface of RARalpha fusion partners is a critical element in APL pathogenesis while pointing to other features of PML for enhancing penetrance and progression.
Assuntos
Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Animais , Medula Óssea/patologia , Carcinógenos/metabolismo , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Regulação para Baixo/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Células Mieloides/metabolismo , Células Mieloides/patologia , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Receptores de Citocinas/metabolismo , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/metabolismo , Transcrição Gênica/genéticaRESUMO
BACKGROUND: In patients with previously untreated metastatic colorectal cancer (mCRC), we conducted a phase 1b/randomized phase 2 trial to define the safety, tolerability, and efficacy of mFOLFOX6 plus bevacizumab (mFOLFOX6/bev) with conatumumab, an investigational, fully human monoclonal IgG1 antibody that specifically activates death receptor 5 (DR5). METHODS: Twelve patients were enrolled in a phase 1b open-label dose-escalation trial of conatumumab with mFOLFOX6/bev; thereafter, 190 patients were randomized 1:1:1 to receive mFOLFOX6/bev in combination with 2 mg/kg conatumumab, 10 mg/kg conatumumab, or placebo. Therapy cycles were repeated every 2 weeks until disease progression or the occurrence of unacceptable toxicity. RESULTS: In phase 1b, conatumumab with mFOLFOX6/bev was tolerated without apparent added toxicity over mFOLFOX6/bev alone. In phase 2, conatumumab with mFOLFOX6/bev did not confer a benefit in progression-free survival when compared with placebo with mFOLFOX6/bev. Toxicity was similar in all treatment arms. Following treatment, similar increases in circulating caspase-3 levels were observed in all arms. CONCLUSIONS: Conatumumab with mFOLFOX6/bev did not offer improved efficacy over the same chemotherapy with placebo in first-line treatment of patients with mCRC. These data do not support further development of conatumumab in advanced CRC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab , Biomarcadores Tumorais/metabolismo , Caspase 3/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/efeitos adversos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
OBJECTIVE: Upon colon cancer metastasis resection in liver, disease outcome is heterogeneous, ranging from indolent to very aggressive, with early recurrence. The aim of this study is to investigate the capability of metastasis associated in colon cancer 1 (MACC1) levels measured in liver metastasis specimens to predict further recurrence of the disease. METHODS: Gene expression and gene dosage of MACC1, hepatocyte growth factor (HGF), and hepatocyte growth factor receptor (MET) were assessed using quantitative realtime polymerase chain reaction on a cohort of 64 liver metastasis samples from patients with complete follow-up of 36 months and detailed clinical annotation. The most relevant mutations associated to prognosis in colorectal cancer, KRAS, and PIK3CA were assessed on the same specimens with Sanger sequencing. RESULTS: Receiver operating characteristic (ROC) analysis revealed that MACC1 mRNA abundance is a good indicator of metastatic recurrence (AUC = 0.65, P < 0.05), whereas no such results were obtained with MET and HGF, nor with gene dosage. Generation of MACC1-based risk classes was capable of successfully separating patients into poor and good prognosis subgroups [hazard ratio (HR) = 5.236, 95% confidence interval (CI) = 1.2068-22.715, P < 0.05]. Also KRAS mutation was significantly associated with higher risk of recurrence (HR = 2.07, 95% CI = 1.048-4.09, P < 0.05). Cox regression multivariate analysis supported the independence of MACC1, but not KRAS, from known prognostic clinical information (Node Size HR = 3.155, 95% CI = 1.4418-6.905, P < 0.001, Preoperative carcinoembryonic antigen HR = 2.359, 95% CI = 1.0203-5.452, P < 0.05, MACC1 HR = 7.2739, 95% CI = 1.6584-31.905, P < 0.01). CONCLUSIONS: MACC1, a new easily detectable biomarker in cancer, is an independent prognostic factor of recurrence after liver resection of colorectal cancer metastasis.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Fatores de Transcrição/genética , Idoso , Biomarcadores Tumorais/genética , Antígeno Carcinoembrionário/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Diagnóstico por Imagem , Feminino , Fator de Crescimento de Hepatócito/genética , Humanos , Masculino , Método de Monte Carlo , Mutação , Terapia Neoadjuvante , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro , Curva ROC , Transativadores , Proteínas ras/genéticaRESUMO
The MET oncogene is amplified in a fraction of human gastric carcinoma cell lines, with consequent overexpression and constitutive activation of the corresponding protein product, the Met tyrosine kinase receptor. This genetically driven hyperactivation of Met is necessary for cancer cell growth and survival, so that Met pharmacological blockade results in cell-cycle arrest or apoptosis (oncogene addiction). MET gene amplification also occurs in vivo in a number of human gastric carcinomas, and clinical trials are now ongoing to assess the therapeutic efficacy of Met inhibitors in this type of malignancy. The aim of our study was to identify a preclinical algorithm of soluble surrogate biomarkers indicative of response to Met inhibition in gastric tumors, as a potential tool to integrate imaging criteria during patient follow-up. We started from a survey of candidate molecules based on antibody proteomics and gene expression profiling; after ELISA validation and analytical quantification, four biomarkers were identified that appeared to be strongly and consistently modulated by Met inhibition in a panel of Met-addicted gastric carcinoma cell lines, but not in Met-independent cell lines. Pharmacologic blockade of Met using specific small-molecule inhibitors led to reduced secretion of IL-8, GROα and the soluble form of uPAR and to increased production of IL-6 both in vitro (in culture supernatants) and in vivo (in the plasma of xenografted mice). If confirmed in patients, this information might prove useful to monitor clinical response to Met-targeted therapies in MET-amplified gastric carcinomas.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Quimiocina CXCL1/sangue , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Indóis/farmacologia , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/sangue , Interleucina-8/genética , Interleucina-8/metabolismo , Lectinas de Ligação a Manose/sangue , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Proteômica , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Neoplasias Gástricas/sangue , Neoplasias Gástricas/genética , Sulfonas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Diabetes-associated long-term hyperglycaemia leads to oxidative stress-mediated fibrosis in different tissues and organs. Endothelial-to-mesenchymal-transition (EndMT) appears to play a role in diabetes-associated fibrotic conditions. Here, we investigate whether EndMT is implicated in the diabetic retinopathy fibrotic process and evaluate the possibility that resveratrol could counteract EndMT by inhibiting high glucose (HG)-induced increases in ROS. Primary Human Retinal Endothelial Cells (HRECs) were either pre-treated for 24 h with 1 µM resveratrol or left untreated, then glucose (30 mM) was applied at 3-day intervals for 10 days. qRT-PCR and ELISA were used to detect mRNA or protein expression of endothelial markers (CD31, CDH5, vWF) or mesenchymal markers (VIM, αSMA and collagen I), respectively. Intracellular ROS levels were measured with carboxy-DCFDA, while NOX-associated ROS levels were evaluated using the NADPH-specific redox biosensor p47-roGFP. Treatment of HRECs with HG increased intracellular ROS levels and promoted phenotype shifting towards EndMT, evidenced by decreased expression of endothelial markers concomitant with increased expression of mesenchymal ones. HG-induced EndMT appears to be mediated by NADPH-associated ROS generation as pre-treatment of HRECs with resveratrol or the NADPH inhibitor, diphenyleneiodonium chloride (DPI), attenuated ROS production and EndMT transition, suggesting that the effect of resveratrol on HG-induced ROS occurs via down-regulation of NADPH oxidase. It is worth noting that resveratrol or Chelerythrine, a Protein kinase C (PKC) inhibitor, reduce ROS and EndMT in HG-exposed cells, suggesting that NADPH activation occurs via a PKC-dependent mechanism. Taken together, our results provide the basis for a resveratrol-based potential protective therapy to prevent diabetic-associated complications.
RESUMO
Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.
Assuntos
Variações do Número de Cópias de DNA/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento do ExomaRESUMO
The initiation of mammalian puberty and the maintenance of female reproductive cycles are events controlled by hypothalamic neurons that secrete the decapeptide gonadotropin-releasing hormone (GnRH). GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The hierarchical control of the process is unknown, but it requires coordinated regulation of these cell-cell interactions. Here we report the functional characterization of a gene (termed enhanced at puberty 1 [EAP1]) that appears to act as an upstream transcriptional regulator of neuronal networks controlling female reproductive function. EAP1 expression increased selectively at puberty in both the nonhuman primate and rodent hypothalamus. EAP1 encoded a nuclear protein expressed in neurons involved in the inhibitory and facilitatory control of reproduction. EAP1 transactivated genes required for reproductive function, such as GNRH1, and repressed inhibitory genes, such as preproenkephalin. It contained a RING finger domain of the C3HC4 subclass required for this dual transcriptional activity. Inhibition of EAP1 expression, targeted to the rodent hypothalamus via lentivirus-mediated delivery of EAP1 siRNAs, delayed puberty, disrupted estrous cyclicity, and resulted in ovarian abnormalities. These results suggest that EAP1 is a transcriptional regulator that, acting within the neuroendocrine brain, contributes to controlling female reproductive function.
Assuntos
Ciclo Estral/metabolismo , Hormônio Liberador de Gonadotropina/biossíntese , Sistema Hipotálamo-Hipofisário/metabolismo , Proteínas de Neoplasias/biossíntese , Neurônios/metabolismo , Precursores de Proteínas/biossíntese , Maturidade Sexual , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação para Baixo/genética , Ciclo Estral/genética , Feminino , Hormônio Liberador de Gonadotropina/genética , Humanos , Sistema Hipotálamo-Hipofisário/citologia , Lentivirus , Macaca mulatta , Proteínas de Neoplasias/genética , Neuroglia/citologia , Neuroglia/patologia , Neurônios/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Precursores de Proteínas/genética , Estrutura Terciária de Proteína/genética , Ratos , Ratos Sprague-Dawley , Securina , Maturidade Sexual/genética , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
Non-human primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.
Assuntos
Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus/genética , Primatas/genética , Primatas/metabolismo , Animais , HumanosRESUMO
Fasting triggers a series of hormonal cues that promote energy balance by inducing glucose output and lipid breakdown in the liver. In response to pancreatic glucagon and adrenal cortisol, the cAMP-responsive transcription factor CREB activates gluconeogenic and fatty acid oxidation programmes by stimulating expression of the nuclear hormone receptor coactivator PGC-1 (refs 2-5). In parallel, fasting also suppresses lipid storage and synthesis (lipogenic) pathways, but the underlying mechanism is unknown. Here we show that mice deficient in CREB activity have a fatty liver phenotype and display elevated expression of the nuclear hormone receptor PPAR-gamma, a key regulator of lipogenic genes. CREB inhibits hepatic PPAR-gamma expression in the fasted state by stimulating the expression of the Hairy Enhancer of Split (HES-1) gene, a transcriptional repressor that is shown here to be a mediator of fasting lipid metabolism in vivo. The coordinate induction of PGC-1 and repression of PPAR-gamma by CREB during fasting provides a molecular rationale for the antagonism between insulin and counter-regulatory hormones, and indicates a potential role for CREB antagonists as therapeutic agents in enhancing insulin sensitivity in the liver.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Jejum , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Triglicerídeos/metabolismoRESUMO
Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.
Assuntos
Antirreumáticos , Artrite Reumatoide , Sinoviócitos , Animais , Antirreumáticos/uso terapêutico , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.
Assuntos
Neoplasias Colorretais , Fosfatidilinositol 3-Quinases , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB , Humanos , Recidiva Local de Neoplasia , Neoplasia Residual , Celulas de Paneth , FenótipoRESUMO
Macrophage-stimulating protein (MSP) exerts proliferative and antiapoptotic effects, suggesting that it may play a role in tubular regeneration after acute kidney injury. In this study, elevated plasma levels of MSP were found both in critically ill patients with acute renal failure and in recipients of renal allografts during the first week after transplantation. In addition, MSP and its receptor, RON, were markedly upregulated in the regenerative phase after glycerol-induced tubular injury in mice. In vitro, MSP stimulated tubular epithelial cell proliferation and conferred resistance to cisplatin-induced apoptosis by inhibiting caspase activation and modulating Fas, mitochondrial proteins, Akt, and extracellular signal-regulated kinase. MSP also enhanced migration, scattering, branching morphogenesis, tubulogenesis, and mesenchymal de-differentiation of surviving tubular cells. In addition, MSP induced an embryonic phenotype characterized by Pax-2 expression. In conclusion, MSP is upregulated during the regeneration of injured tubular cells, and it exerts multiple biologic effects that may aid recovery from acute kidney injury.
Assuntos
Injúria Renal Aguda/sangue , Fator de Crescimento de Hepatócito/sangue , Transplante de Rim , Túbulos Renais/fisiologia , Proteínas Proto-Oncogênicas/sangue , Receptores Proteína Tirosina Quinases/sangue , Regeneração/fisiologia , Idoso , Animais , Estudos de Casos e Controles , Técnicas de Cultura de Células , Sobrevivência Celular , Estado Terminal , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
Purpose: c-KIT overexpression is well recognized in cancers such as gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), melanoma, non-small cell lung cancer (NSCLC), and acute myelogenous leukemia (AML). Treatment with the small-molecule inhibitors imatinib, sunitinib, and regorafenib resulted in resistance (c-KIT mutant tumors) or limited activity (c-KIT wild-type tumors). We selected an anti-c-KIT ADC approach to evaluate the anticancer activity in multiple disease models.Experimental Design: A humanized anti-c-KIT antibody LMJ729 was conjugated to the microtubule destabilizing maytansinoid, DM1, via a noncleavable linker (SMCC). The activity of the resulting ADC, LOP628, was evaluated in vitro against GIST, SCLC, and AML models and in vivo against GIST and SCLC models.Results: LOP628 exhibited potent antiproliferative activity on c-KIT-positive cell lines, whereas LMJ729 displayed little to no effect. At exposures predicted to be clinically achievable, LOP628 demonstrated single administration regressions or stasis in GIST and SCLC xenograft models in mice. LOP628 also displayed superior efficacy in an imatinib-resistant GIST model. Further, LOP628 was well tolerated in monkeys with an adequate therapeutic index several fold above efficacious exposures. Safety findings were consistent with the pharmacodynamic effect of neutropenia due to c-KIT-directed targeting. Additional toxicities were considered off-target and were consistent with DM1, such as effects in the liver and hematopoietic/lymphatic system.Conclusions: The preclinical findings suggest that the c-KIT-directed ADC may be a promising therapeutic for the treatment of mutant and wild-type c-KIT-positive cancers and supported the clinical evaluation of LOP628 in GIST, AML, and SCLC patients. Clin Cancer Res; 24(17); 4297-308. ©2018 AACR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Xenoenxertos , Humanos , Mesilato de Imatinib/farmacologia , Imunoconjugados/imunologia , Camundongos , Mutação , Neoplasias/classificação , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/imunologiaRESUMO
Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-ß pathway activity, epithelial-mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances.
Assuntos
Neoplasias Colorretais/classificação , Neoplasias Colorretais/genética , Células Estromais/metabolismo , Transcriptoma , Animais , Antineoplásicos Imunológicos/farmacologia , Linhagem da Célula , Cetuximab/farmacologia , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Perfilação da Expressão Gênica , Genes p53 , Glicólise , Xenoenxertos , Humanos , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos , Instabilidade de Microssatélites , Mutação , Prognóstico , Transdução de Sinais , Células Estromais/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The adult mammalian spinal cord contains neural stem and/or progenitor cells that slowly multiply throughout life and differentiate exclusively into glia. The contribution of adult progenitors to repair has been highlighted in recent studies, demonstrating extensive cell proliferation and gliogenesis following central nervous system (CNS) trauma. The present experiments aimed to determine the relative roles of endogenously dividing progenitor cells versus quiescent progenitor cells in posttraumatic gliogenesis. Using the mitotic indicator bromodeoxyuridine (BrdU) and a retroviral vector, we found that, in the adult female Fisher 344 rat, endogenously dividing neural progenitors are acutely vulnerable in response to T8 dorsal hemisection spinal cord injury. We then studied the population of cells that divide postinjury in the injury epicenter by delivering BrdU or retrovirus at 24 hours after spinal cord injury. Animals were euthanized at five timepoints postinjury, ranging from 6 hours to 9 weeks after BrdU delivery. At all timepoints, we observed extensive proliferation of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells. BrdU+ incorporation was noted to be prominent in NG2-immunoreactive progenitors that matured into oligodendrocytes, and in a transient population of microglia. Using a green fluorescence protein (GFP) hematopoietic chimeric mouse, we determined that 90% of the dividing cells in this early proliferation event originate from the spinal cord, whereas only 10% originate from the bone marrow. Our results suggest that dividing, NG2-expressing progenitor cells are vulnerable to injury, but a separate, immature population of neural stem and/or progenitor cells is activated by injury and rapidly divides to replace this vulnerable population.
Assuntos
Linhagem da Célula/fisiologia , Regeneração Nervosa/fisiologia , Neuroglia/citologia , Traumatismos da Medula Espinal/patologia , Células-Tronco/citologia , Cicatrização/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos F344 , Medula Espinal/citologia , Medula Espinal/fisiologia , Células-Tronco/classificação , Fatores de TempoRESUMO
We studied telomere length in 32 CML patients who discontinued imatinib after achieving complete molecular remission and 32 age-sex-matched controls. The relative telomere length (RTL) was determined by q-PCR as the telomere to single copy gene (36B4) ratio normalized to a reference sample (K-562 DNA). Age-corrected RTL (acRTL) was also obtained. The 36-month probability of treatment-free remission (TFR) was 59.4 %. TFR patients showed shorter acRTL compared to relapsed (mean ± SD = 0.01 ± 0.14 vs 0.20 ± 0.21; p = 0.01). TFR was significantly higher in CML patients with acRTL ≤0.09 (78.9 vs 30.8 %, p = 0.002). CML stem cells harboring longer telomeres possibly maintain a proliferative potential after treatment discontinuation.