RESUMO
The activation and accumulation of lung fibroblasts resulting in aberrant deposition of extracellular matrix components, is a pathogenic hallmark of Idiopathic Pulmonary Fibrosis, a lethal and incurable disease. In this report, increased expression of TKS5, a scaffold protein essential for the formation of podosomes, was detected in the lung tissue of Idiopathic Pulmonary Fibrosis patients and bleomycin-treated mice. Τhe profibrotic milieu is found to induce TKS5 expression and the formation of prominent podosome rosettes in lung fibroblasts, that are retained ex vivo, culminating in increased extracellular matrix invasion. Tks5+/- mice are found resistant to bleomycin-induced pulmonary fibrosis, largely attributed to diminished podosome formation in fibroblasts and decreased extracellular matrix invasion. As computationally predicted, inhibition of src kinase is shown to potently attenuate podosome formation in lung fibroblasts and extracellular matrix invasion, and bleomycin-induced pulmonary fibrosis, suggesting pharmacological targeting of podosomes as a very promising therapeutic option in pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Podossomos , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transporte Vesicular , Bleomicina , Matriz Extracelular , Fibroblastos , Fibrose Pulmonar Idiopática/induzido quimicamente , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismoRESUMO
BACKGROUND: Expression quantitative trait loci (eQTL) studies provide insights into regulatory mechanisms underlying disease risk. Expanding studies of gene regulation to underexplored populations and to medically relevant tissues offers potential to reveal yet unknown regulatory variants and to better understand disease mechanisms. Here, we performed eQTL mapping in subcutaneous (S) and visceral (V) adipose tissue from 106 Greek individuals (Greek Metabolic study, GM) and compared our findings to those from the Genotype-Tissue Expression (GTEx) resource. RESULTS: We identified 1,930 and 1,515 eGenes in S and V respectively, over 13% of which are not observed in GTEx adipose tissue, and that do not arise due to different ancestry. We report additional context-specific regulatory effects in genes of clinical interest (e.g. oncogene ST7) and in genes regulating responses to environmental stimuli (e.g. MIR21, SNX33). We suggest that a fraction of the reported differences across populations is due to environmental effects on gene expression, driving context-specific eQTLs, and suggest that environmental effects can determine the penetrance of disease variants thus shaping disease risk. We report that over half of GM eQTLs colocalize with GWAS SNPs and of these colocalizations 41% are not detected in GTEx. We also highlight the clinical relevance of S adipose tissue by revealing that inflammatory processes are upregulated in individuals with obesity, not only in V, but also in S tissue. CONCLUSIONS: By focusing on an understudied population, our results provide further candidate genes for investigation regarding their role in adipose tissue biology and their contribution to disease risk and pathogenesis.
Assuntos
Predisposição Genética para Doença , Locos de Características Quantitativas , Humanos , Grécia , Regulação da Expressão Gênica , Genótipo , Polimorfismo de Nucleotídeo Único , Estudo de Associação Genômica Ampla/métodosRESUMO
Pathogenesis and progression of lung cancer are governed by complex interactions between the environment and host genetic susceptibility, which is further modulated by genetic and epigenetic changes. Autotaxin (ATX, ENPP2) is a secreted glycoprotein that catalyzes the extracellular production of lysophosphatidic acid (LPA), a growth-factor-like phospholipid that is further regulated by phospholipid phosphatases (PLPP). LPA's pleiotropic effects in almost all cell types are mediated through at least six G-protein coupled LPA receptors (LPAR) that exhibit overlapping specificities, widespread distribution, and differential expression profiles. Here we use both preclinical models of lung cancer and clinical samples (from patients and healthy controls) to investigate the expression levels, activity, and biological role of the above components of the ATX/LPA axis in lung cancer. ENPP2 was genetically altered in 8% of patients with lung cancer, whereas increased ATX staining and activity were detected in patient biopsies and sera, respectively. Moreover, PLPP3 expression was consistently downregulated in patients with lung cancer. Comparable observations were made in the two most widely used animal models of lung cancer, the carcinogen urethane-induced and the genetically engineered K-rasG12D -driven models, where genetic deletion of Enpp2 or Lpar1 resulted in disease attenuation, thus confirming a procarcinogenic role of LPA signaling in the lung. Expression profiling data analysis suggested that metabolic rewiring may be implicated in the procarcinogenic effects of the ATX/LPA axis in K-ras- G12D -driven lung cancer pathogenesis.Significance: These findings establish the role of ATX/LPA in lung carcinogenesis, thus expanding the mechanistic links between pulmonary fibrosis and cancer. Cancer Res; 78(13); 3634-44. ©2018 AACR.
Assuntos
Carcinogênese/patologia , Neoplasias Pulmonares/patologia , Lisofosfolipídeos/metabolismo , Fosfatidato Fosfatase/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Idoso , Animais , Conjuntos de Dados como Assunto , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Diester Fosfórico Hidrolases/genética , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Uretana/toxicidadeRESUMO
Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.
Assuntos
Adenocarcinoma/genética , Antineoplásicos/farmacologia , Neoplasias Pulmonares/genética , Células Mieloides/patologia , Derrame Pleural Maligno/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Galinhas , Membrana Corioalantoide , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Cavidade Pleural/citologia , Cavidade Pleural/patologia , Derrame Pleural Maligno/tratamento farmacológico , Derrame Pleural Maligno/patologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/metabolismo , Baço/citologia , Baço/patologia , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Autotaxin (ATX) is a secreted lysophospholipase D that catalyzes the production of lysophosphatidic acid (LPA), a pleiotropic growth-factor-like lysophospholipid. Increased ATX expression has been detected in various chronic inflammatory disorders and different types of cancer; however, little is known about its role and mode of action in liver fibrosis and cancer. Here, increased ATX expression was detected in chronic liver disease (CLD) patients of different etiologies, associated with shorter overall survival. In mice, different hepatotoxic stimuli linked with the development of different forms of CLDs were shown to stimulate hepatocyte ATX expression, leading to increased LPA levels, activation of hepatic stellate cells (HSCs), and amplification of profibrotic signals. Hepatocyte-specific, conditional genetic deletion and/or transgenic overexpression of ATX established a liver profibrotic role for ATX/LPA, whereas pharmacological ATX inhibition studies suggested ATX as a possible therapeutic target in CLDs. In addition, hepatocyte ATX ablation and the consequent deregulation of lipid homeostasis was also shown to attenuate hepatocellular carcinoma (HCC) development, thus implicating ATX/LPA in the causative link of cirrhosis and HCC. CONCLUSION: ATX is a novel player in the pathogenesis of liver fibrosis and cancer and a promising therapeutic target. (Hepatology 2017;65:1369-1383).
Assuntos
Benzoxazóis/farmacologia , Carcinoma Hepatocelular/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Diester Fosfórico Hidrolases/genética , Piperazinas/farmacologia , Animais , Biópsia por Agulha , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Diester Fosfórico Hidrolases/efeitos dos fármacosRESUMO
The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/ß-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/ß-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/ß-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Intestinos/patologia , RNA Longo não Codificante/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Longo não Codificante/genética , Via de Sinalização Wnt/genéticaRESUMO
Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1ß, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.
Assuntos
Mastócitos/metabolismo , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Mesilato de Imatinib , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/dietoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Piperazinas/farmacologia , Cavidade Pleural/metabolismo , Cavidade Pleural/patologia , Derrame Pleural Maligno/tratamento farmacológico , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Triptases/genética , Triptases/metabolismoRESUMO
HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuR's requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuR's mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuR's absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.
Assuntos
Antígenos de Superfície/metabolismo , Morfogênese , Placenta , Proteínas de Ligação a RNA/metabolismo , Animais , Antígenos de Superfície/genética , Células Cultivadas , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Anormalidades Musculoesqueléticas/genética , Anormalidades Musculoesqueléticas/patologia , Fenótipo , Placenta/anatomia & histologia , Placenta/embriologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Baço/anormalidades , Baço/embriologiaRESUMO
RATIONALE: Despite intense research efforts, the etiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. OBJECTIVES: To discover novel genes and/or cellular pathways involved in the pathogenesis of the disease. METHODS: We performed expression profiling of disease progression in a well-characterized animal model of the disease. Differentially expressed genes that were identified were compared with all publicly available expression profiles both from human patients and animal models. The role of hypoxia-inducible factor (HIF)-1alpha in disease pathogenesis was examined with a series of immunostainings, both in the animal model as well as in tissue microarrays containing tissue samples of human patients, followed by computerized image analysis. MEASUREMENTS AND MAIN RESULTS: Comparative expression profiling produced a prioritized gene list of high statistical significance, which consisted of the most likely disease modifiers identified so far in pulmonary fibrosis. Extending beyond target identification, a series of meta-analyses produced a number of biological hypotheses on disease pathogenesis. Among them, the role of HIF-1 signaling was further explored to reveal HIF-1alpha overexpression in the hyperplastic epithelium of fibrotic lungs, colocalized with its target genes p53 and Vegf. CONCLUSIONS: Comparative expression profiling was shown to be a highly efficient method in identifying deregulated genes and pathways. Moreover, tissue microarrays and computerized image analysis allowed for the high-throughput and unbiased assessment of histopathologic sections, adding substantial confidence in pathologic evaluations. More importantly, our results suggest an early primary role of HIF-1 in alveolar epithelial cell homeostasis and disease pathogenesis, provide insights on the pathophysiologic differences of different interstitial pneumonias, and indicate the importance of assessing the efficacy of pharmacologic inhibitors of HIF-1 activity in the treatment of pulmonary fibrosis.
Assuntos
Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/fisiopatologia , Animais , Apoptose , Progressão da Doença , Epitélio/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Pneumonia/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transdução de Sinais , Distribuição Tecidual , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems, resulting in various pathological disorders. Idiopathic Pulmonary Fibrosis is a prototype fibrotic disease involving abnormal wound healing in response to multiple sites of ongoing alveolar epithelial injury. METHODOLOGY/PRINCIPAL FINDINGS: To decipher the role of TNF and TNF-mediated inflammation in the development of fibrosis, we have utilized the bleomycin-induced animal model of Pulmonary Fibrosis and a series of genetically modified mice lacking components of TNF signaling. Transmembrane TNF expression is shown to be sufficient to elicit an inflammatory response, but inadequate for the transition to the fibrotic phase of the disease. Soluble TNF expression is shown to be crucial for lymphocyte recruitment, a prerequisite for TGF-b1 expression and the development of fibrotic lesions. Moreover, through a series of bone marrow transfers, the necessary TNF expression is shown to originate from the non-hematopoietic compartment further localized in apoptosing epithelial cells. CONCLUSIONS: These results suggest a primary detrimental role of soluble TNF in the pathologic cascade, separating it from the beneficial role of transmembrane TNF, and indicate the importance of assessing the efficacy of soluble TNF antagonists in the treatment of Idiopathic Pulmonary Fibrosis.
Assuntos
Inflamação/etiologia , Inflamação/metabolismo , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Bleomicina/toxicidade , Transplante de Medula Óssea , Modelos Animais de Doenças , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/etiologia , Inflamação/induzido quimicamente , Inflamação/patologia , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais , Solubilidade , Fator de Crescimento Transformador beta1/biossíntese , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology-assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease.