RESUMO
Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor derived from parafollicular thyroid gland cells. In both hereditary MTC and sporadic forms, genetic changes result in fundamental changes, and prognosis and mutational status are highly correlated. In this work, biomarker genes (DEGs and DEmiRNAs) for MTC will be computationally identified in order to help in their diagnosis and treatment. The gene expression profiles of two different types of studies, namely without-treatment (wo-trt) and with-treatment (w-trt), are considered for discovering biomarkers. The datasets were retrieved from the GEO database, and the DEGs and DEmiRNAs were analyzed using ExpressAnalyst and GEO2R. The functional analysis of DEGs and DEmiRNAs was performed, and most of the pathways enriched related to thyroid oncological pathways such as MAPK pathway,mTOR pathway, and PI3K-AKT Signaling pathway. Through this conclusion, the RET gene was upregulated wo-trt; the dinaciclib treatment RET gene was down-regulated computationally. To optimize the therapeutic targeting of RET, greater research into the mechanisms regulating RET transcription is necessary.
Assuntos
Biomarcadores Tumorais , Carcinoma Neuroendócrino , Biologia Computacional , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/tratamento farmacológico , Carcinoma Neuroendócrino/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
The damage to the mechanical barrier of the intestinal mucosa is the initiating factor and the core link of the progression of ulcerative colitis (UC). Protecting the mechanical barrier of the intestinal mucosa is of great significance for improving the health status of UC patients. ZO-1 is a key scaffold protein of the mechanical barrier of the intestinal mucosa, and its fusion with the membrane of the intestinal epithelium is a necessary condition to maintain the integrity of the mechanical barrier of the intestinal mucosa. Enteric glial cells (EGCs) play an important role in the maintenance of intestinal homeostasis and have become a new target for regulating intestinal health in recent years. In this study, we found that glycyrol (GC), a representative coumarin compound isolated from Licorice (Glycyrrhiza uralensis Fisch, used for medicine and food), can alleviate UC by promoting the production of neurotrophic factor GDNF in mice EGCs. Specifically, we demonstrated that GC promotes the production of GDNF, then activates its receptor RET, promotes ZO-1 fusion with cell membranes, and protects the intestinal mucosal mechanical barrier. The results of this study can provide new ideas for the prevention and treatment of UC.
Assuntos
Colite Ulcerativa , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Mucosa Intestinal , Neuroglia , Proteína da Zônula de Oclusão-1 , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Camundongos , Humanos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Masculino , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Camundongos Endogâmicos C57BL , Cumarínicos/farmacologia , Cumarínicos/química , Transdução de Sinais/efeitos dos fármacos , Glycyrrhiza/químicaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is among the strongest dopamine neuron function- and survival-promoting factors known. Due to this reason, it has clinical relevance in dopamine disorders such as Parkinson's disease and schizophrenia. In the striatum, GDNF is exclusively expressed in interneurons, which make up only about 0.6% of striatal cells. Despite clinical significance, histological analysis of striatal GDNF system arborization and relevance to incoming dopamine axons, which bear its receptor RET, has remained enigmatic. This is mainly due to the lack of antibodies able to visualize GDNF- and RET-positive cellular processes; here, we overcome this problem by using knock-in marker alleles. We find that GDNF neurons chemoattract RET+ axons at least seven times farther in distance than medium spiny neurons (MSNs), which make up 95% of striatal neurons. Furthermore, we provide evidence that tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, is enriched towards GDNF neurons in the dopamine axons. Finally, we find that GDNF neuron arborizations occupy approximately only twelve times less striatal volume than 135 times more abundant MSNs. Collectively, our results improve our understanding of how endogenous GDNF affects striatal dopamine system function.
Assuntos
Axônios , Corpo Estriado , Neurônios Dopaminérgicos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteínas Proto-Oncogênicas c-ret , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Axônios/metabolismo , Corpo Estriado/metabolismo , Corpo Estriado/citologia , Camundongos , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Neurônios Dopaminérgicos/metabolismo , Dopamina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios Espinhosos MédiosRESUMO
Tyrosine kinase (TK) fusions are frequently found in cancers, either as initiating events or as a mechanism of resistance to targeted therapy. Partner genes and exons in most TK fusions are followed typical recurrent patterns, but the underlying mechanisms and clinical implications of these patterns are poorly understood. By developing Functionally Active Chromosomal Translocation Sequencing (FACTS), we discover that typical TK fusions involving ALK, ROS1, RET and NTRK1 are selected from pools of chromosomal rearrangements by two major determinants: active transcription of the fusion partner genes and protein stability. In contrast, atypical TK fusions that are rarely seen in patients showed reduced protein stability, decreased downstream oncogenic signaling, and were less responsive to inhibition. Consistently, patients with atypical TK fusions were associated with a reduced response to TKI therapies. Our findings highlight the principles of oncogenic TK fusion formation and selection in cancers, with clinical implications for guiding targeted therapy.
Assuntos
Neoplasias , Proteínas de Fusão Oncogênica , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-ret , Translocação Genética , Humanos , Neoplasias/genética , Neoplasias/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Linhagem Celular TumoralRESUMO
Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases.
Assuntos
Proteína Adaptadora GRB2 , Sistema de Sinalização das MAP Quinases , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas c-ret , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteínas ras , Humanos , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/genética , Células HEK293 , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosforilação , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genéticaRESUMO
RET fusion is an oncogenic driver in 1-2 % of patients with non-small cell lung cancer (NSCLC). Although RET-positive tumors have been treated with multikinase inhibitors such as vandetanib or RET-selective inhibitors, ultimately resistance to them develops. Here we established vandetanib resistance (VR) clones from LC-2/ad cells harboring CCDC6-RET fusion and explored the molecular mechanism of the resistance. Each VR clone had a distinct phenotype, implying they had acquired resistance via different mechanisms. Consistently, whole exome-seq and RNA-seq revealed that the VR clones had unique mutational signatures and expression profiles, and shared only a few common remarkable events. AXL and IGF-1R were activated as bypass pathway in different VR clones, and sensitive to a combination of RET and AXL inhibitors or IGF-1R inhibitors, respectively. SMARCA4 loss was also found in a particular VR clone and 55 % of post-TKI lung tumor tissues, being correlated with higher sensitivity to SMARCA4/SMARCA2 dual inhibition and shorter PFS after subsequent treatments. Finally, we detected an increased number of damaged mitochondria in one VR clone, which conferred sensitivity to mitochondrial electron transfer chain inhibitors. Increased mitochondria were also observed in post-TKI biopsy specimens in 13/20 cases of NSCLC, suggesting a potential strategy targeting mitochondria to treat resistant tumors. Our data propose new promising therapeutic options to combat resistance to RET inhibitors in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , Mitocôndrias , Piperidinas , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-ret , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Helicases/antagonistas & inibidores , Proteínas do CitoesqueletoRESUMO
Aberrant RET kinase signaling is activated in numerous cancers including lung, thyroid, breast, pancreatic, and prostate. Recent approvals of selective RET inhibitors, pralsetinib and selpercatinib, has shifted the focus of RET kinase drug discovery programs towards the development of selective inhibitors. However, selective inhibitors invariably lose efficacy as the selective nature of the inhibitor places Darwinian-like pressure on the tumor to bypass treatment through the selection of novel oncogenic drivers. Further, selective inhibitors are restricted for use in tumors with specific genetic backgrounds that do not encompass diverse patient classes. Here we report the identification of a pyrimido indole RET inhibitor found to also have activity against TRK. This selective dual RET/TRK inhibitor can be utilized in tumors with both RET and TRK genetic backgrounds and can also provide blockade of NTRK-fusions that are selected for from RET inhibitor treatments. Efforts towards developing dual RET/TRK inhibitors can be beneficial in terms of encompassing more diverse patient classes while also achieving blockade against emerging resistance mechanisms.
Assuntos
Indóis , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-ret , Receptor trkA , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Descoberta de Drogas , Indóis/química , Indóis/farmacologia , Indóis/síntese química , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/síntese química , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Relação Estrutura-AtividadeRESUMO
Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.
Assuntos
Membrana Celular , Proteínas de Membrana , Proteínas Proto-Oncogênicas c-ret , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Membrana Celular/metabolismo , Transdução de Sinais , Transporte Proteico , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proliferação de Células , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologiaRESUMO
Gestational diabetes mellitus (GDM) presents a substantial population health concern. Previous studies have revealed that GDM can ultimately influence nephron endowment. In this study, we established a GDM mouse model to investigate the embryological alterations and molecular mechanisms underlying the development of congenital anomalies of the kidney and urinary tract (CAKUT) affected by GDM. Our study highlights that GDM could contribute to the manifestation of CAKUT, with prevalent phenotypes characterized by isolated hydronephrosis and duplex kidney complicated with hydronephrosis in mice. Ectopic ureteric buds (UBs) and extended length of common nephric ducts (CNDs) were noted in the metanephric development stage. The expression of Ret and downstream p-ERK activity were enhanced in UBs, which indicated the alteration of RET/MAPK/ERK pathway may be one of the mechanisms contributing to the increased occurrence of CAKUT associated with GDM.
Assuntos
Diabetes Gestacional , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-ret , Anormalidades Urogenitais , Refluxo Vesicoureteral , Animais , Feminino , Camundongos , Gravidez , Diabetes Gestacional/metabolismo , Rim/anormalidades , Rim/metabolismo , Rim/embriologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Sistema Urinário/anormalidades , Sistema Urinário/embriologia , Anormalidades Urogenitais/etiologia , Anormalidades Urogenitais/genética , Anormalidades Urogenitais/patologiaAssuntos
Motilidade Gastrointestinal , Mucosa Intestinal , Proteínas Proto-Oncogênicas c-ret , Transdução de Sinais , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Motilidade Gastrointestinal/fisiologia , Mucosa Intestinal/metabolismo , Humanos , Animais , CamundongosRESUMO
Activation of RET tyrosine kinase plays a critical role in the pathogenesis of various cancers, including non-small cell lung cancer, papillary thyroid cancers, multiple endocrine neoplasia type 2A and 2B (MEN2A, MEN2B), and familial medullary thyroid cancer. Gene fusions and point mutations in the RET proto-oncogene result in constitutive activation of RET signaling pathways. Consequently, developing effective inhibitors to target RET is of utmost importance. Small molecules have shown promise as inhibitors by binding to the kinase domain of RET and blocking its enzymatic activity. However, the emergence of resistance due to single amino acid changes poses a significant challenge. In this study, a structure-based dynamic pharmacophore-driven approach using E-pharmacophore modeling from molecular dynamics trajectories is proposed to select low-energy favorable hypotheses, and ML-trained QSAR models to predict pIC50 values of compounds. For this aim, extensive small molecule libraries were screened using developed ligand-based models, and potent compounds that are capable of inhibiting RET activation were proposed.
Assuntos
Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Relação Quantitativa Estrutura-Atividade , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Humanos , Estrutura Molecular , Descoberta de Drogas , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , FarmacóforoRESUMO
BACKGROUND: HSCR is a complex genetic disorder characterized by the absence of ganglion cells in the intestine, leading to a functional obstruction. It is due to a disruption of complex signaling pathways within the gene regulatory network (GRN) during the development of the enteric nervous system (ENS), including SRY-Box Transcription Factor 10 (SOX10) and REarranged during Transfection (RET). This study evaluated the expressions of SOX10 and RET in HSCR patients in Indonesia. METHODS: Total RNA of 19 HSCR ganglionic and aganglionic colons and 16 control colons were analyzed using quantitative real-time polymerase chain reaction for SOX10 and RET with GAPDH as the reference gene. Livak's method (2-ΔΔCT) was used to determine the expression levels of SOX10 and RET. RESULTS: Most patients were males (68.4%), in the short aganglionosis segment (78.9%), and had undergone transanal endorectal pull-through (36.6%). There were significant upregulated SOX10 expressions in both ganglionic (2.84-fold) and aganglionic (3.72-fold) colon of HSCR patients compared to controls' colon (ΔCT 5.21 ± 2.04 vs. 6.71 ± 1.90; p = 0.032; and ΔCT 4.82 ± 1.59 vs. 6.71 ± 1.90; p = 0.003; respectively). Interestingly, the RET expressions were significantly downregulated in both ganglionic (11.71-fold) and aganglionic (29.96-fold) colon of HSCR patients compared to controls' colon (ΔCT 12.54 ± 2.21 vs. 8.99 ± 3.13; p = 0.0004; and ΔCT 13.90 ± 2.64 vs. 8.99 ± 3.13; p = 0.0001; respectively). CONCLUSIONS: Our study shows aberrant SOX10 and RET expressions in HSCR patients, implying the critical role of SOX10 and RET in the pathogenesis of HSCR, particularly in the Indonesian population. Our study further confirms the involvement of SOX10-RET within the GNR during the ENS development.
Assuntos
Doença de Hirschsprung , Masculino , Humanos , Feminino , Doença de Hirschsprung/metabolismo , Transdução de Sinais , Indonésia , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Fatores de Transcrição SOXE/genéticaRESUMO
INTRODUCTION: Previous studies have suggested that the tyrosine kinase receptor RET plays a significant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in human iPSC-derived hematopoiesis has not been tested so far. METHODS: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. The hematopoietic potential was tested using 2D cultures and evaluated regarding the phenotype and the clonogenic potential of generated cells. RESULTS: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of primitive hematopoietic cells (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific activated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. CONCLUSION: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related to MAPK activation.
Assuntos
Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Receptores Proteína Tirosina Quinases/metabolismo , Diferenciação Celular/genética , Hematopoese/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
Our previous study showed that glial-derived neurotrophic factor (GDNF) expression is upregulated in asthmatic human lungs, and GDNF regulates calcium responses through its receptor GDNF family receptor α1 (GFRα1) and RET receptor in human airway smooth muscle (ASM) cells. In this study, we tested the hypothesis that airway GDNF contributes to airway hyperreactivity (AHR) and remodeling using a mixed allergen mouse model. Adult C57BL/6J mice were intranasally exposed to mixed allergens (ovalbumin, Aspergillus, Alternaria, house dust mite) over 4 wk with concurrent exposure to recombinant GDNF, or extracellular GDNF chelator GFRα1-Fc. Airway resistance and compliance to methacholine were assessed using FlexiVent. Lung expression of GDNF, GFRα1, RET, collagen, and fibronectin was examined by RT-PCR and histology staining. Allergen exposure increased GDNF expression in bronchial airways including ASM and epithelium. Laser capture microdissection of the ASM layer showed increased mRNA for GDNF, GFRα1, and RET in allergen-treated mice. Allergen exposure increased protein expression of GDNF and RET, but not GFRα1, in ASM. Intranasal administration of GDNF enhanced baseline responses to methacholine but did not consistently potentiate allergen effects. GDNF also induced airway thickening, and collagen deposition in bronchial airways. Chelation of GDNF by GFRα1-Fc attenuated allergen-induced AHR and particularly remodeling. These data suggest that locally produced GDNF, potentially derived from epithelium and/or ASM, contributes to AHR and remodeling relevant to asthma.NEW & NOTEWORTHY Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.
Assuntos
Asma , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Animais , Camundongos , Alérgenos , Colágeno , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Cloreto de Metacolina/farmacologia , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRα1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRα1 complex also supports synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRα1 assembly determined by crystallography and electron microscopy, revealing two GFRα1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly between adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRα1:GFRα1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-driven cell adhesion, its relationship to trophic signalling, and the central role played by GFRα1.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteínas Proto-Oncogênicas c-ret , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. RESEARCH DESIGN AND METHODS: We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from 'real-life' small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. RESULTS: The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. CONCLUSIONS: Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Inclusão em Parafina , Humanos , Quinase do Linfoma Anaplásico/análise , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Proteínas de Fusão Oncogênica/análise , Proteínas Tirosina Quinases/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ret/análise , Proteínas Proto-Oncogênicas c-ret/metabolismo , RNA , Imunoquímica/métodosRESUMO
The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.
Assuntos
Neoplasias das Glândulas Suprarrenais , Feocromocitoma , Humanos , Animais , Camundongos , Feocromocitoma/genética , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Mutação em Linhagem Germinativa , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Mutação/genética , Ubiquitinação , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
The RET (REarranged during Transfection) gene, which encodes for a transmembrane receptor tyrosine kinase, is an established oncogene associated with the etiology and progression of multiple types of cancer. Oncogenic RET mutations and rearrangements resulting in gene fusions have been identified in many adult cancers, including medullary and papillary thyroid cancers, lung adenocarcinomas, colon and breast cancers, and many others. While genetic RET aberrations are much less common in pediatric solid tumors, increased RET expression has been shown to be associated with poor prognosis in children with solid tumors such as neuroblastoma, prompting an interest in RET inhibition as a form of therapy for these children. A number of kinase inhibitors currently in use for patients with cancer have RET inhibitory activity, but these inhibitors also display activity against other kinases, resulting in unwanted side effects and limiting their safety and efficacy. Recent efforts have been focused on developing more specific RET inhibitors, but due to high levels of conservation between kinase binding pockets, specificity remains a drug design challenge. Here, we review the background of RET as a potential therapeutic target in neuroblastoma tumors and the results of recent preclinical studies and clinical trials evaluating the safety and efficacy of RET inhibition in adults and children. We also present a novel approach to drug discovery leveraging the chemical phenomenon of atropisomerism to develop specific RET inhibitors and present preliminary data demonstrating the efficacy of a novel RET inhibitor against neuroblastoma tumor cells.
Assuntos
Neoplasias Pulmonares , Neuroblastoma , Adulto , Criança , Humanos , Desenho de Fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.