RESUMO
Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.
Assuntos
Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Imunoterapia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/imunologia , Animais , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Carcinogênese/imunologia , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/imunologia , Progressão da Doença , Humanos , Fígado/imunologia , Fígado/patologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.
Assuntos
Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/agonistas , Imunidade Adaptativa , Envelhecimento/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Enfisema/metabolismo , Feminino , Humanos , Imunidade Inata , Pulmão/metabolismo , Receptor beta de Linfotoxina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Microbiota , Hepatopatia Gordurosa não Alcoólica , Humanos , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/complicações , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Camundongos Endogâmicos C57BL , Fígado/patologia , Fibrose , Cirrose Hepática/complicações , Camundongos Transgênicos , Imunoglobulina A/metabolismo , Imunoglobulina A/farmacologia , Modelos Animais de Doenças , Dieta Hiperlipídica/efeitos adversosRESUMO
Fibrosis is a condition characterized by the excessive accumulation of extracellular matrix proteins in tissues, leading to organ dysfunction and failure. Recent studies have identified EP300, a histone acetyltransferase, as a crucial regulator of the epigenetic changes that contribute to fibrosis. In fact, EP300-mediated acetylation of histones alters global chromatin structure and gene expression, promoting the development and progression of fibrosis. Here, we review the role of EP300-mediated epigenetic regulation in multi-organ fibrosis and its potential as a therapeutic target. We discuss the preclinical evidence that suggests that EP300 inhibition can attenuate fibrosis-related molecular processes, including extracellular matrix deposition, inflammation, and epithelial-to-mesenchymal transition. We also highlight the contributions of small molecule inhibitors and gene therapy approaches targeting EP300 as novel therapies against fibrosis.
Assuntos
Epigênese Genética , Histonas , Humanos , Fibrose , Histonas/metabolismo , Matriz Extracelular/metabolismo , Histona Acetiltransferases/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismoRESUMO
BACKGROUND & AIMS: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver-resident macrophages. However, hepatocytes, the parenchymal cells of the liver, also possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct antiviral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IkkßΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/ß signaling-(IfnarΔHep), or interferon-α/ß signaling in myeloid cells-(IfnarΔMyel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1 signaling but rather on Toll-like receptor signaling. LCMV-infected IkkßΔHep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKß, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IkkßΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IfnarΔHep mice, whereas IfnarΔMyel mice were able to control LCMV infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/ß-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/ß signaling, which is pivotal for strong early ISG responses, immune cell infiltration and hepatic viral clearance. LAY SUMMARY: Innate immune cells have been ascribed a primary role in controlling viral clearance upon hepatic infections. We identified a novel dual role for NF-κB signaling in infected hepatocytes which was crucial for maximizing interferon responses and initiating adaptive immunity, thereby efficiently controlling hepatic virus replication.
Assuntos
Hepacivirus/genética , Hepatite C Crônica/genética , Hepatite C Crônica/imunologia , Hepatócitos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Subunidade p50 de NF-kappa B/genética , Polimorfismo de Nucleotídeo Único , Fator de Transcrição RelA/metabolismo , Replicação Viral/genética , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Genótipo , Hepatite C Crônica/virologia , Humanos , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Coriomeningite Linfocítica/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Adulto JovemRESUMO
The respiratory epithelium arises from alveolar epithelial progenitors which differentiate into alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. AT2 cells are stem cells in the lung critical for the repair process after injury. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report that the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using miR-142 KO mice, we demonstrate an increase in the AT2/AT1 cell number ratio. Overexpression of miR-142 in alveolar progenitor cells in vivo led to the opposite effect. Examination of the KO lungs at E18.5 revealed enhanced expression of miR-142 targets Apc, Ep300 and Kras associated with increased ß-catenin and p-Erk signaling. Silencing of miR-142 expression in lung explants grown in vitro triggers enhanced Sftpc expression as well as increased AT2/AT1 cell number ratio. Pharmacological inhibition of Ep300-ß-catenin but not Erk in vitro prevented the increase in Sftpc expression triggered by loss of miR-142. These results suggest that the glucocorticoid-miR-142-Ep300-ß-catenin signaling axis controls pneumocyte maturation.
Assuntos
Células Epiteliais Alveolares/citologia , Linhagem da Célula , Pulmão/crescimento & desenvolvimento , MicroRNAs/genética , Organogênese , Mucosa Respiratória/citologia , Células Epiteliais Alveolares/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Mucosa Respiratória/fisiologiaRESUMO
RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.
Assuntos
Transformação Celular Neoplásica/metabolismo , Endorribonucleases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular , Movimento Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Endorribonucleases/química , Estabilidade Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Camundongos , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2(-/-)). RESULTS: We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2(-/-) mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression. CONCLUSIONS: Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.
Assuntos
Proteína HMGA2/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Via de Sinalização Wnt , Animais , Diferenciação Celular , Proliferação de Células , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Receptores Frizzled/metabolismo , Fator de Transcrição GATA6/metabolismo , Proteína HMGA2/deficiência , Camundongos , Camundongos Knockout , FenótipoRESUMO
Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.
Assuntos
Antígenos CD18/genética , Células Epiteliais/metabolismo , Cadeias beta de Integrinas/genética , Proteoma/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética , Animais , Antígenos CD18/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Células Epiteliais/citologia , Regulação da Expressão Gênica , Cadeias beta de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anotação de Sequência Molecular , Ligação Proteica , Proteína C/genética , Proteína C/metabolismo , Proteoma/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Análise Serial de TecidosRESUMO
BACKGROUND: Lung cancer (LC) causes more deaths worldwide than any other cancer type. Despite advances in therapeutic strategies, the fatality rate of LC cases remains high (95%) since the majority of patients are diagnosed at late stages when patient prognosis is poor. Analysis of the International Association for the Study of Lung Cancer (IASLC) database indicates that early diagnosis is significantly associated with favorable outcome. However, since symptoms of LC at early stages are unspecific and resemble those of benign pathologies, current diagnostic approaches are mostly initiated at advanced LC stages. METHODS: We developed a LC diagnosis test based on the analysis of distinct RNA isoforms expressed from the GATA6 and NKX2-1 gene loci, which are detected in exhaled breath condensates (EBCs). Levels of these transcript isoforms in EBCs were combined to calculate a diagnostic score (the LC score). In the present study, we aimed to confirm the applicability of the LC score for the diagnosis of early stage LC under clinical settings. Thus, we evaluated EBCs from patients with early stage, resectable non-small cell lung cancer (NSCLC), who were prospectively enrolled in the EMoLung study at three sites in Germany. RESULTS: LC score-based classification of EBCs confirmed its performance under clinical conditions, achieving a sensitivity of 95.7%, 91.3% and 84.6% for LC detection at stages I, II and III, respectively. CONCLUSIONS: The LC score is an accurate and non-invasive option for early LC diagnosis and a valuable complement to LC screening procedures based on computed tomography.
RESUMO
Background: Small cell lung cancer (SCLC) is an extremely aggressive cancer type with a patient median survival of 6-12 months. Epidermal growth factor (EGF) signaling plays an important role in triggering SCLC. In addition, growth factor-dependent signals and alpha-, beta-integrin (ITGA, ITGB) heterodimer receptors functionally cooperate and integrate their signaling pathways. However, the precise role of integrins in EGF receptor (EGFR) activation in SCLC remains elusive. Methods: We analyzed human precision-cut lung slices (hPCLS), retrospectively collected human lung tissue samples and cell lines by classical methods of molecular biology and biochemistry. In addition, we performed RNA-sequencing-based transcriptomic analysis in human lung cancer cells and human lung tissue samples, as well as high-resolution mass spectrometric analysis of the protein cargo from extracellular vesicles (EVs) that were isolated from human lung cancer cells. Results: Our results demonstrate that non-canonical ITGB2 signaling activates EGFR and RAS/MAPK/ERK signaling in SCLC. Further, we identified a novel SCLC gene expression signature consisting of 93 transcripts that were induced by ITGB2, which may be used for stratification of SCLC patients and prognosis prediction of LC patients. We also found a cell-cell communication mechanism based on EVs containing ITGB2, which were secreted by SCLC cells and induced in control human lung tissue RAS/MAPK/ERK signaling and SCLC markers. Conclusions: We uncovered a mechanism of ITGB2-mediated EGFR activation in SCLC that explains EGFR-inhibitor resistance independently of EGFR mutations, suggesting the development of therapies targeting ITGB2 for patients with this extremely aggressive lung cancer type.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Estudos Retrospectivos , Receptores ErbB/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Integrinas/genética , MutaçãoRESUMO
Single-cell transcriptomics (scRNA-seq) has revolutionized the understanding of the spatial architecture of tissue structure and function. Advancing the "transcript-centric" view of scRNA-seq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyze post-translational modifications. Here, by combining spatial cell sorting with transcriptomics and quantitative proteomics/phosphoproteomics, we established the spatially resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signaling mechanisms. Phosphorylation of receptor tyrosine kinases was detected preferentially in the central vein area, resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation identified Tie receptor signaling as a selective and specific regulator of vascular Wnt activity orchestrating angiocrine signaling, thereby controlling hepatocyte function during liver regeneration. Taken together, the study has yielded fundamental insight into the spatial organization of liver endothelial cell signaling. Spatial sorting may be employed as a universally adaptable strategy for multiomic analyses of scRNA-seq-defined cellular (sub)-populations.
Assuntos
Regeneração Hepática/genética , Fígado/crescimento & desenvolvimento , Fosfoproteínas/genética , Transcriptoma/genética , Células Endoteliais/metabolismo , Endotélio/crescimento & desenvolvimento , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Fosforilação/genética , Proteômica/métodos , RNA-Seq , Regeneração/genética , Análise de Célula Única , Via de Sinalização Wnt/genéticaRESUMO
In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.
Assuntos
Desmetilação do DNA , Nucleossomos/metabolismo , Iniciação da Transcrição Genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/química , Cromatina/metabolismo , Células HEK293 , Proteína HMGA2/metabolismo , Histonas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Camundongos , Fosforilação , Fosfosserina/metabolismo , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição , Ativação Transcricional/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Although NF-κB is known to play a pivotal role in lung cancer, contributing to tumor growth, microenvironmental changes, and metastasis, the epigenetic regulation of NF-κB in tumor context is largely unknown. Here we report that the IKK2/NF-κB signaling pathway modulates metastasis-associated protein 2 (MTA2), a component of the nucleosome remodeling and deacetylase complex (NuRD). In triple transgenic mice, downregulation of IKK2 (Sftpc-cRaf-IKK2DN) in cRaf-induced tumors in alveolar epithelial type II cells restricted tumor formation, whereas activation of IKK2 (Sftpc-cRaf-IKK2CA) supported tumor growth; both effects were accompanied by altered expression of MTA2. Further studies employing genetic inhibition of MTA2 suggested that in primary tumor growth, independent of IKK2, MTA2/NuRD corepressor complex negatively regulates NF-κB signaling and tumor growth, whereas later dissociation of MTA2/NuRD complex from the promoter of NF-κB target genes and IKK2-dependent positive regulation of MTA2 leads to activation of NF-κB signaling, epithelial-mesenchymal transition, and lung tumor metastasis. These findings reveal a previously unrecognized biphasic role of MTA2 in IKK2/NF-κB-driven primary-to-metastatic lung tumor progression. Addressing the interaction between MTA2 and NF-κB would provide potential targets for intervention of tumor growth and metastasis. SIGNIFICANCE: These findings strongly suggest a prominent role of MTA2 in primary tumor growth, lung metastasis, and NF-κB signaling modulatory functions.
Assuntos
Histona Desacetilases/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Histona Desacetilases/genética , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Inflamação/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/genética , Proteínas Repressoras/genética , Transdução de Sinais , Transativadores/genética , Microambiente TumoralRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and highly lethal lung disease with unknown etiology and poor prognosis. IPF patients die within 2 years after diagnosis mostly due to respiratory failure. Current treatments against IPF aim to ameliorate patient symptoms and to delay disease progression. Unfortunately, therapies targeting the causes of or reverting IPF have not yet been developed. Here we show that reduced levels of miRNA lethal 7d (MIRLET7D) in IPF compromise epigenetic gene silencing mediated by the ribonucleoprotein complex MiCEE. In addition, we find that hyperactive EP300 reduces nuclear HDAC activity and interferes with MiCEE function in IPF. Remarkably, EP300 inhibition reduces fibrotic hallmarks of in vitro (patient-derived primary fibroblast), in vivo (bleomycin mouse model), and ex vivo (precision-cut lung slices, PCLS) IPF models. Our work provides the molecular basis for therapies against IPF using EP300 inhibition.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Histona Desacetilase 1/metabolismo , Fibrose Pulmonar Idiopática/patologia , MicroRNAs/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Bleomicina/toxicidade , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Proteína p300 Associada a E1A/antagonistas & inibidores , Fibroblastos , Inativação Gênica , Histona Desacetilase 2/metabolismo , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Cultura Primária de Células , Ribonucleoproteínas/genéticaRESUMO
The majority of the eukaryotic genome is transcribed into noncoding RNAs (ncRNAs), which are important regulators of different nuclear processes by controlling chromatin structure. However, the full extent of ncRNA function has remained elusive. Here we deciphered the function of the microRNA Mirlet7d as a key regulator of bidirectionally transcribed genes. We found that nuclear Mirlet7d binds ncRNAs expressed from these genes. Mirlet7d-ncRNA duplexes are further bound by C1D, which in turn targets the RNA exosome complex and the polycomb repressive complex 2 (PRC2) to the bidirectionally active loci. The exosome degrades the ncRNAs, whereas PRC2 induces heterochromatin and transcriptional silencing through EZH2. Moreover, this multicomponent RNA-protein complex, which we named MiCEE, tethers the regulated genes to the perinucleolar region and thus is required for proper nucleolar organization. Our study demonstrates that the MiCEE complex mediates epigenetic silencing of bidirectionally expressed genes and global genome organization.
Assuntos
Nucléolo Celular/genética , Região Organizadora do Nucléolo/genética , RNA não Traduzido/genética , Animais , Linhagem Celular , Nucléolo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Drosophila , Epigênese Genética , Exossomos , Inativação Gênica , Células HEK293 , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Região Organizadora do Nucléolo/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA não Traduzido/metabolismo , Transcrição GênicaRESUMO
Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Early LC diagnosis is crucial to reduce the high case fatality rate of this disease. In this case-control study, we developed an accurate LC diagnosis test using retrospectively collected formalin-fixed paraffin-embedded (FFPE) human lung tissues and prospectively collected exhaled breath condensates (EBCs). Following international guidelines for diagnostic methods with clinical application, reproducible standard operating procedures (SOP) were established for every step comprising our LC diagnosis method. We analyzed the expression of distinct mRNAs expressed from GATA6 and NKX2-1, key regulators of lung development. The Em/Ad expression ratios of GATA6 and NKX2-1 detected in EBCs were combined using linear kernel support vector machines (SVM) into the LC score, which can be used for LC detection. LC score-based diagnosis achieved a high performance in an independent validation cohort. We propose our method as a non-invasive, accurate, and low-price option to complement the success of computed tomography imaging (CT) and chest X-ray (CXR) for LC diagnosis.