RESUMO
The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.
RESUMO
Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although several Notch inhibitors are under development as cancer therapies, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins that mimic the tension-generating mechanism of endogenous ligands. SNAGs were designed by fusing a high-affinity variant of the Notch ligand Delta-like 4 (DLL4) to antibody fragments that induce target internalization. This bispecific format enables the SNAG-bound biomarkers to "pull" on Notch receptors, triggering Notch activation in mixed populations of biomarker-expressing and non-expressing cells. SNAGs targeting the immune checkpoint PDL1 potently activated Notch in co-cultures of Notch1- and PDL1-expressing cells, but not in monocultures of Notch1-expressing cells alone. Additional SNAGs targeting the tumor antigens CD19 and HER2 also activated Notch in mixed cell populations, indicating that the SNAG design concept is adaptable to multiple biomarkers. SNAG-mediated Notch activation was blocked by a dynamin inhibitor, and efficacy increased dramatically when SNAGs were dimerized via fusion to antibody Fc domains, suggesting that endocytosis and multimerization are important for optimal SNAG function. These insights will greatly expand our ability to modulate Notch signaling for applications in immunotherapy and regenerative medicine.
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Pathogenic variants in the JAG1 gene are a primary cause of the multi-system disorder Alagille syndrome. Although variant detection rates are high for this disease, there is uncertainty associated with the classification of missense variants that leads to reduced diagnostic yield. Consequently, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications. We generated a library of 2,832 JAG1 nucleotide variants within exons 1-7, a region with a high number of reported missense variants, and designed a high-throughput assay to measure JAG1 membrane expression, a requirement for normal function. After calibration using a set of 175 known or predicted pathogenic and benign variants included within the variant library, 486 variants were characterized as functionally abnormal (n = 277 abnormal and n = 209 likely abnormal), of which 439 (90.3%) were missense. We identified divergent membrane expression occurring at specific residues, indicating that loss of the wild-type residue itself does not drive pathogenicity, a finding supported by structural modeling data and with broad implications for clinical variant classification both for Alagille syndrome and globally across other disease genes. Of 144 uncertain variants reported in patients undergoing clinical or research testing, 27 had functionally abnormal membrane expression, and inclusion of our data resulted in the reclassification of 26 to likely pathogenic. Functional evidence augments the classification of genomic variants, reducing uncertainty and improving diagnostics. Inclusion of this repository of functional evidence during JAG1 variant reclassification will significantly affect resolution of variant pathogenicity, making a critical impact on the molecular diagnosis of Alagille syndrome.
Assuntos
Síndrome de Alagille , Proteína Jagged-1 , Mutação de Sentido Incorreto , Síndrome de Alagille/genética , Proteína Jagged-1/genética , Humanos , Éxons/genéticaRESUMO
The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (â¼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismoRESUMO
The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.
Assuntos
Receptores Notch , Transdução de Sinais , Receptores Notch/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
The Notch pathway regulates a diverse array of cell fate decisions, making it an enticing target in cancer therapy and regenerative medicine. During the early stages of Notch drug development, off-target toxicity precluded the approval of Notch inhibitors for the treatment of cancer. However, recent advances in our understanding of Notch structure and signaling have led to the development of several innovative Notch-based biotechnologies. In addition to new classes of inhibitors, pharmacological Notch activators have been shown to enhance osteogenesis and various aspects of T cell function. Furthermore, the mechanosensitive negative regulatory region (NRR) of the Notch receptor has been converted into synthetic Notch (synNotch) receptors with fully customizable signaling circuits. We review emergent Notch-based compounds, biologics, and cell therapies while highlighting the challenges and opportunities they face on the path to clinical development.
Assuntos
Neoplasias , Receptores Notch , Humanos , Receptores Notch/metabolismo , Receptores Notch/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais/fisiologia , BiotecnologiaRESUMO
The atypical cadherins Fat and Dachsous are key regulators of cell growth and animal development. In contrast to classical cadherins, which form homophilic interactions to segregate cells, Fat and Dachsous cadherins form heterophilic interactions to induce cell polarity within tissues. Here, we determine the co-crystal structure of the human homologs Fat4 and Dachsous1 (Dchs1) to establish the molecular basis for Fat-Dachsous interactions. The binding domains of Fat4 and Dchs1 form an extended interface along extracellular cadherin (EC) domains 1-4 of each protein. Biophysical measurements indicate that Fat4-Dchs1 affinity is among the highest reported for cadherin superfamily members, which is attributed to an extensive network of salt bridges not present in structurally similar protocadherin homodimers. Furthermore, modeling suggests that unusual extracellular phosphorylation modifications directly modulate Fat-Dachsous binding by introducing charged contacts across the interface. Collectively, our analyses reveal how the molecular architecture of Fat4-Dchs1 enables them to form long-range, high-affinity interactions to maintain planar cell polarity.
Assuntos
Caderinas , Polaridade Celular , Proteínas Supressoras de Tumor , Humanos , Caderinas/química , Proteínas Supressoras de Tumor/química , Proteínas Relacionadas a Caderinas/químicaRESUMO
Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.
Assuntos
Fucose , Melanoma , Humanos , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/metabolismo , Fucose/metabolismo , Melanoma/tratamento farmacológico , Imunoterapia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologiaRESUMO
The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, binds human and murine Notch receptors with 500- to 1,000-fold increased affinity compared with wild-type human Delta-like 4. DeltaMAX also potently activates Notch in plate-bound, bead-bound and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch in reporter and neuronal differentiation assays, highlighting its dual utility as an agonist or antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in T cells. Taken together, our data define DeltaMAX as a versatile tool for broad-spectrum activation or inhibition of Notch signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Peptídeos e Proteínas de Sinalização Intercelular , Humanos , Animais , Camundongos , Ligantes , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Transdução de Sinais/fisiologia , Receptores Notch/metabolismoRESUMO
The Notch pathway converts receptor-ligand interactions at the cell surface into a transcriptional response in the receiver cell. In recent years, synthetic Notch systems (synNotch) that respond to different inputs and transduce different transcriptional responses have been engineered. One class of synNotch systems uses antibody-antigen interactions at the cell surface to induce the proteolytic cleavage cascade of the endogenous Notch autoregulatory core and the consequent release of a synNotch intracellular domain (ICD), converting surface antigen detection into a cellular response. While the activation of endogenous Notch requires ubiquitylation and subsequent endocytosis of the ligand ICD, these synNotch systems do not seem to have such a requirement because the synNotch ligands completely lack an ICD. This observation raises questions about existing models for the synNotch activation mechanism. Here, we test how different structural and biochemical factors affect the dependence of endogenous and synthetic Notch activation on ligand ICD. We compare the behavior of antibody-antigen synNotch (aa-synNotch) to that of endogenous Notch, and to a synNotch system that uses rapamycin induced dimerization of FK506 binding protein (FKBP) and FKBP rapamycin binding (FRB) domaindimerization domains (ff-synNotch), which still requires a ligand ICD. We found that differences in receptor-ligand affinity, in the identity of the transmembrane domain, or in the presence or absence of extracellular epidermal growth factor repeats cannot explain the differences in ligand ICD requirement that distinguishes aa-synNotch from endogenous Notch or ff-synNotch. We also found that unlike endogenous Notch and ff-synNotch, the aa-synNotch system does not exhibit trans-endocytosis of the receptor extracellular domain into the sender cell. These findings suggest that the aa-synNotch systems bypass the ligand ICD requirement because antigen-antibody pairs are able to promote other adhesive cell-cell interactions that provide the mechanical tension needed for ligand activation.
Assuntos
Fator de Crescimento Epidérmico , Transdução de Sinais , Ligantes , Proteínas de Ligação a Tacrolimo , Sirolimo , Antígenos de SuperfícieRESUMO
The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.
Assuntos
Antígenos CD/metabolismo , Ativação Linfocitária , Animais , Anticorpos , Fibrinogênio , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoterapia , Ligantes , Camundongos , Receptores Imunológicos , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Aberrant activation of Wnt/ß-catenin signaling is strongly associated with many diseases including cancer invasion and metastasis. Small-molecule targeting of the central signaling node of this pathway, ß-catenin, is a biologically rational approach to abolish hyperactivation of ß-catenin signaling but has been demonstrated to be a difficult task. Herein, we report a drug-like small molecule, ZW4864, that binds with ß-catenin and selectively disrupts the protein-protein interaction (PPI) between B-cell lymphoma 9 (BCL9) and ß-catenin while sparing the ß-catenin/E-cadherin PPI. ZW4864 dose-dependently suppresses ß-catenin signaling activation, downregulates oncogenic ß-catenin target genes, and abrogates invasiveness of ß-catenin-dependent cancer cells. More importantly, ZW4864 shows good pharmacokinetic properties and effectively suppresses ß-catenin target gene expression in the patient-derived xenograft mouse model. This study offers a selective chemical probe to explore ß-catenin-related biology and a drug-like small-molecule ß-catenin/BCL9 disruptor for future drug development.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Piperidinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos SCID , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/farmacocinética , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.
Assuntos
DNA/metabolismo , Células Dendríticas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Citoplasma/metabolismo , Endocitose/fisiologia , Feminino , Células HEK293 , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.
Assuntos
Antígenos CD/imunologia , Butirofilinas/antagonistas & inibidores , Butirofilinas/imunologia , Linfócitos Intraepiteliais/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/genética , Butirofilinas/genética , Feminino , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.
Assuntos
Ácidos e Sais Biliares/metabolismo , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Ácidos e Sais Biliares/química , Sítios de Ligação/efeitos dos fármacos , Ácidos Cólicos/química , Ácidos Cólicos/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Especificidade por SubstratoRESUMO
Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/ß-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.
Assuntos
Colo/crescimento & desenvolvimento , Anticorpos de Cadeia Única/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt , Sítios de Ligação , Colo/metabolismo , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/imunologiaRESUMO
Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.
Assuntos
Família de Proteínas EGF/metabolismo , Fucose/análogos & derivados , Fucose/farmacologia , Fucosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Fucose/química , Fucose/genética , Fucosiltransferases/genética , Células HEK293 , Humanos , Ligantes , Ligação ProteicaRESUMO
The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Receptores Notch/metabolismo , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Microambiente Tumoral , Animais , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Receptores Notch/agonistas , Receptores Notch/antagonistas & inibidores , Receptores Notch/deficiência , Proteínas Repressoras/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológicoRESUMO
The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.