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1.
J Biomed Sci ; 28(1): 69, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635096

RESUMO

BACKGROUND: Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. METHODS: Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. RESULTS: Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. CONCLUSIONS: Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.


Assuntos
Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
2.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502405

RESUMO

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system (CNS). MS commonly affects the cerebellum causing acute and chronic symptoms. Cerebellar signs significantly contribute to clinical disability, and symptoms such as tremor, ataxia, and dysarthria are difficult to treat. Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in demyelinating pathologies such as MS. In autopsy tissue from patients with MS, increased expression of FGF1, FGF2, FGF9, and FGFR1 was found in lesion areas. Recent research using mouse models has focused on regions such as the spinal cord, and data on the expression of FGF/FGFR in the cerebellum are not available. In recent EAE studies, we detected that oligodendrocyte-specific deletion of FGFRs results in a milder disease course, less cellular infiltrates, and reduced neurodegeneration in the spinal cord. The objective of this study was to characterize the role of FGFR1 in oligodendrocytes in the cerebellum. Conditional deletion of FGFR1 in oligodendrocytes (Fgfr1ind-/-) was achieved by tamoxifen application, EAE was induced using the MOG35-55 peptide. The cerebellum was analyzed by histology, immunohistochemistry, and western blot. At day 62 p.i., Fgfr1ind-/- mice showed less myelin and axonal degeneration compared to FGFR1-competent mice. Infiltration of CD3(+) T cells, Mac3(+) cells, B220(+) B cells and IgG(+) plasma cells in cerebellar white matter lesions (WML) was less in Fgfr1ind-/-mice. There were no effects on the number of OPC or mature oligodendrocytes in white matter lesion (WML). Expression of FGF2 and FGF9 associated with less myelin and axonal degeneration, and of the pro-inflammatory cytokines IL-1ß, IL-6, and CD200 was downregulated in Fgfr1ind-/- mice. The FGF/FGFR signaling protein pAkt, BDNF, and TrkB were increased in Fgfr1ind-/- mice. These data suggest that cell-specific deletion of FGFR1 in oligodendrocytes has anti-inflammatory and neuroprotective effects in the cerebellum in the EAE disease model of MS.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Oligodendroglia/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Doenças Desmielinizantes/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/fisiopatologia , Fatores de Crescimento de Fibroblastos/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/metabolismo , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/farmacologia , Oligodendroglia/patologia , Fosforilação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismo , Substância Branca/metabolismo
3.
J Biol Chem ; 297(4): 101149, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34473994

RESUMO

Metabolic flexibility is the capacity of cells to alter fuel metabolism in response to changes in metabolic demand or nutrient availability. It is critical for maintaining cellular bioenergetics and is involved in the pathogenesis of cardiovascular disease and metabolic disorders. However, the regulation and function of metabolic flexibility in lymphatic endothelial cells (LECs) remain unclear. We have previously shown that glycolysis is the predominant metabolic pathway to generate ATP in LECs and that fibroblast growth factor receptor (FGFR) signaling controls lymphatic vessel formation by promoting glycolysis. Here, we found that chemical inhibition of FGFR activity or knockdown of FGFR1 induces substantial upregulation of fatty acid ß-oxidation (FAO) while reducing glycolysis and cellular ATP generation in LECs. Interestingly, such compensatory elevation was not observed in glucose oxidation and glutamine oxidation. Mechanistic studies show that FGFR blockade promotes the expression of carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme of FAO; this is achieved by dampened extracellular signal-regulated protein kinase activation, which in turn upregulates the expression of the peroxisome proliferator-activated receptor alpha. Metabolic analysis further demonstrates that CPT1A depletion decreases total cellular ATP levels in FGFR1-deficient rather than wildtype LECs. This result suggests that FAO, which makes a negligible contribution to cellular energy under normal conditions, can partially compensate for energy deficiency caused by FGFR inhibition. Consequently, CPT1A silencing potentiates the effect of FGFR1 knockdown on impeding LEC proliferation and migration. Collectively, our study identified a key role of metabolic flexibility in modulating the effect of FGFR signaling on LEC growth.


Assuntos
Proliferação de Células , Células Endoteliais/metabolismo , Glicólise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Técnicas de Silenciamento de Genes , Humanos , PPAR alfa/genética , PPAR alfa/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
4.
EMBO Mol Med ; 13(9): e13193, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34369083

RESUMO

KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically targeting KRAS-driven tumors remain elusive. Here, we show that combined treatment with fibroblast growth factor receptor 1 (FGFR1) and polo-like kinase 1 (PLK1) inhibitors evoke synergistic cytotoxicity in KRAS-mutant tumor models in vitro and in vivo. Pharmacological and genetic suppression of FGFR1 and PLK1 synergizes to enhance anti-proliferative effects and cell death in KRAS-mutant lung and pancreatic but not colon nor KRAS wild-type cancer cells. Mechanistically, co-targeting FGFR1 and PLK1 upregulates reactive oxygen species (ROS), leading to oxidative stress-activated c-Jun N-terminal kinase (JNK)/p38 pathway and E2F1-induced apoptosis. We further delineate that autophagy protects from PLK1/FGFR1 inhibitor cytotoxicity and that antagonizing the compensation mechanism by clinically approved chloroquine fully realizes the therapeutic potential of PLK1 and FGFR1 targeting therapy, producing potent and durable responses in KRAS-mutant patient-derived xenografts and a genetically engineered mouse model of Kras-induced lung adenocarcinoma. These results suggest a previously unappreciated role for FGFR1 and PLK1 in the surveillance of metabolic stress and demonstrate a synergistic drug combination for treating KRAS-mutant cancer.


Assuntos
Neoplasias Pulmonares , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
5.
Biomolecules ; 11(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34439755

RESUMO

FGFRs are cell surface receptors that, when activated by specific FGFs ligands, transmit signals through the plasma membrane, regulating key cellular processes such as differentiation, division, motility, metabolism and death. We have recently shown that the modulation of the spatial distribution of FGFR1 at the cell surface constitutes an additional mechanism for fine-tuning cellular signaling. Depending on the multivalent, engineered ligand used, the clustering of FGFR1 into diverse supramolecular complexes enhances the efficiency and modifies the mechanism of receptor endocytosis, alters FGFR1 lifetime and modifies receptor signaling, ultimately determining cell fate. Here, we present a novel approach to generate multivalent FGFR1 ligands. We functionalized FGF1 for controlled oligomerization by developing N- and C-terminal fusions of FGF1 with the Fc fragment of human IgG1 (FGF1-Fc and Fc-FGF1). As oligomerization scaffolds, we employed GFPpolygons, engineered GFP variants capable of well-ordered multivalent display, fused to protein G to ensure binding of Fc fragment. The presented strategy allows efficient assembly of oligomeric FGFR1 ligands with up to twelve receptor binding sites. We show that multivalent FGFR1 ligands are biologically active and trigger receptor clustering on the cell surface. Importantly, the approach described in this study can be easily adapted to oligomerize alternative growth factors to control the activity of other cell surface receptors.


Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Imunoglobulina G/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Análise por Conglomerados , Endocitose , Endossomos/metabolismo , Proteínas de Fluorescência Verde/química , Humanos , Ligantes , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Fosforilação , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Transdução de Sinais
6.
Cells ; 10(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34360002

RESUMO

Fibrosis is a deleterious invasion of tissues associated with many pathological conditions, such as Duchenne muscular dystrophy (DMD) for which no cure is at present available for its prevention or its treatment. Fibro-adipogenic progenitors (FAPs) are resident cells in the human skeletal muscle and can differentiate into myofibroblasts, which represent the key cell population responsible for fibrosis. In this study, we delineated the pool of microRNAs (miRNAs) that are specifically modulated by TGFß1 in FAPs versus myogenic progenitors (MPs) by a global miRNome analysis. A subset of candidates, including several "FibromiRs", was found differentially expressed between FAPs and MPs and was also deregulated in DMD versus healthy biopsies. Among them, the expression of the TGFß1-induced miR-199a~214 cluster was strongly correlated with the fibrotic score in DMD biopsies. Loss-of-function experiments in FAPs indicated that a miR-214-3p inhibitor efficiently blocked expression of fibrogenic markers in both basal conditions and following TGFß1 stimulation. We found that FGFR1 is a functional target of miR-214-3p, preventing the signaling of the anti-fibrotic FGF2 pathway during FAP fibrogenesis. Overall, our work demonstrates that the « FibromiR ¼ miR-214-3p is a key activator of FAP fibrogenesis by modulating the FGF2/FGFR1/TGFß axis, opening new avenues for the treatment of DMD.


Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , MicroRNAs/genética , Distrofia Muscular de Duchenne/genética , Miofibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta1/genética , Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia/genética , Adolescente , Adulto , Sequência de Bases , Diferenciação Celular , Criança , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibrose , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Miofibroblastos/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Células-Tronco/patologia , Fator de Crescimento Transformador beta1/metabolismo
7.
Cells ; 10(8)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34440670

RESUMO

The heteroreceptor complexes present a novel biological principle for signal integration. These complexes and their allosteric receptor-receptor interactions are bidirectional and novel targets for treatment of CNS diseases including mental diseases. The existence of D2R-5-HT2AR heterocomplexes can help explain the anti-schizophrenic effects of atypical antipsychotic drugs not only based on blockade of 5-HT2AR and of D2R in higher doses but also based on blocking the allosteric enhancement of D2R protomer signaling by 5-HT2AR protomer activation. This research opens a new understanding of the integration of DA and 5-HT signals released from DA and 5-HT nerve terminal networks. The biological principle of forming 5-HT and other heteroreceptor complexes in the brain also help understand the mechanism of action for especially the 5-HT hallucinogens, including putative positive effects of e.g., psilocybin and the indicated prosocial and anti-stress actions of MDMA (ecstasy). The GalR1-GalR2 heterodimer and the putative GalR1-GalR2-5-HT1 heteroreceptor complexes are targets for Galanin N-terminal fragment Gal (1-15), a major modulator of emotional networks in models of mental disease. GPCR-receptor tyrosine kinase (RTK) heteroreceptor complexes can operate through transactivation of FGFR1 via allosteric mechanisms and indirect interactions over GPCR intracellular pathways involving protein kinase Src which produces tyrosine phosphorylation of the RTK. The exciting discovery was made that several antidepressant drugs such as TCAs and SSRIs as well as the fast-acting antidepressant drug ketamine can directly bind to the TrkB receptor and provide a novel mechanism for their antidepressant actions. Understanding the role of astrocytes and their allosteric receptor-receptor interactions in modulating forebrain glutamate synapses with impact on dorsal raphe-forebrain serotonin neurons is also of high relevance for research on major depressive disorder.


Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Neurônios Dopaminérgicos/metabolismo , Transtornos Mentais/metabolismo , Receptor Cross-Talk , Receptor 5-HT2A de Serotonina/metabolismo , Receptores 5-HT1 de Serotonina/metabolismo , Neurônios Serotoninérgicos/metabolismo , Animais , Antidepressivos/uso terapêutico , Antipsicóticos/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/fisiopatologia , Transtornos Mentais/psicologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 2 de Galanina/metabolismo , Receptor 5-HT2A de Serotonina/genética , Receptores de Dopamina D2/metabolismo , Receptores 5-HT1 de Serotonina/genética , Transdução de Sinais
8.
Cell Death Dis ; 12(7): 696, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257270

RESUMO

Trastuzumab resistance negatively influences the clinical efficacy of the therapy for human epidermal growth factor receptor 2 (HER2) positive gastric cancer (GC), and the underlying mechanisms remain elusive. Exploring the mechanisms and finding effective approaches to address trastuzumab resistance are of great necessity. Here, we confirmed that endoplasmic reticulum (ER) stress-induced trastuzumab resistance by up-regulating miR-301a-3p in HER2-positive GC cells. Moreover, we elucidated that miR-301a-3p mediated trastuzumab resistance by down-regulating the expression of leucine-rich repeats and immunoglobulin-like domains containing protein 1 (LRIG1) and subsequently activating the expression of insulin-like growth factor 1 receptor (IGF-1R) and fibroblast growth factor receptor 1 (FGFR1) under ER stress. We also found that intercellular transfer of miR-301a-3p by exosomes disseminated trastuzumab resistance. The present study demonstrated that exosomal miR-301a-3p could serve as a non-invasive biomarker for trastuzumab resistance, which was maybe a novel potential therapeutic target to overcome trastuzumab resistance and improve the curative effect of trastuzumab in HER2-positive GC patients.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , MicroRNAs/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Trastuzumab/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Receptor ErbB-2/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Mol Med Rep ; 23(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34240224

RESUMO

Atopic dermatitis (AD) is a chronic inflammatory skin disease that seriously affects quality of life. Quinine is a bitter taste receptor agonist that exhibits antimalarial effects. The aim of the present study was to examine the therapeutic effects of quinine in AD­like mice. AD was induced with 2,4­dinitrochlorobenzene, and the mice were treated with 10 mg/kg quinine for 1, 4 and 7 days. A total of 60 BALB/c mice were divided into the following groups: Healthy, AD­like, AD­like + quinine and healthy + quinine, with 1, 4 and 7 days groups for each treatment. Blood was extracted from all mice and ELISA was performed to detect immunoglobulin E (IgE) levels. H&E­stained tissue sections were prepared from skin lesions on the backs of the mice and pathological changes were observed. Cytokines were detected via ELISA, and the filaggrin (FLG) and kallikrein­7 (KLK7) proteins were detected via western blotting and immunohistochemistry. IKKα and NF­κB mRNA were analyzed via reverse transcription­quantitative PCR. Quinine ameliorated skin damage in the AD­like mice, reduced IgE expression in the blood, inhibited expression of IKKα and NF­κB, reduced cytokine secretion, reduced KLK7 expression, reduced scratching frequency, increased FLG expression and repaired the skin barrier. These results suggested that quinine exhibited therapeutic effects in AD­like mice.


Assuntos
Dermatite Atópica/tratamento farmacológico , Quinina/farmacologia , Quinina/uso terapêutico , Animais , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Dinitroclorobenzeno/toxicidade , Modelos Animais de Doenças , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imunoglobulina E/sangue , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia
10.
J Med Chem ; 64(15): 10666-10679, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34269576

RESUMO

Aberrant activation of FGFR has been linked to the pathogenesis of many tumor types. Selective inhibition of FGFR has emerged as a promising approach for cancer treatment. Herein, we describe the discovery of compound 38 (INCB054828, pemigatinib), a highly potent and selective inhibitor of FGFR1, FGFR2, and FGFR3 with excellent physiochemical properties and pharmacokinetic profiles. Pemigatinib has received accelerated approval from the U.S. Food and Drug Administration for the treatment of adults with previously treated, unresectable locally advanced or metastatic cholangiocarcinoma with a FGFR2 fusion or other rearrangement. Additional clinical trials are ongoing to evaluate pemigatinib in patients with FGFR alterations.


Assuntos
Descoberta de Drogas , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Estados Unidos , United States Food and Drug Administration
11.
DNA Cell Biol ; 40(8): 1112-1123, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34165368

RESUMO

Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is recognized critical in bone deteriorations of osteoporosis. However, the specific mechanisms that determine the fate of BMSCs remain elusive. MicroRNA-133a (miR-133a), a highly conserved microRNA, was investigated under both in vitro and in vivo conditions. In the in vitro study, cell proliferation, cell apoptosis, and osteoblast/adipocyte differentiation of BMSCs as a result of overexpression or knockdown of miR-133a was investigated. In the in vivo study, the ovariectomy (OVX) model was applied on mice, with further treatment of the models with BMSC-specific miR-133a antagomir through femur intramedullary injection. Microcomputed tomography scanning and histological analysis of the proximal and middle femur were performed to evaluate the morphological changes. The results revealed that overexpression of miR-133a suppressed cell proliferation, cell viability, and osteoblast differentiation of BMSCs, but increased adipocyte differentiation. We also found that FGFR1, an important upstream regulator of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signal pathway, was a major target of miR-133a. We also recorded that BMSC-specific knockdown of miR-133a attenuates bone loss in OVX mice. Our study suggested that miR-133a played an important role in maintaining the viability and balance between osteoblast and adipocyte differentiation of BMSCs through the MAPK/ERK signaling pathway by targeting FGFR1.


Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteoporose Pós-Menopausa/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Antagomirs/uso terapêutico , Células da Medula Óssea/citologia , Sobrevivência Celular , Células Cultivadas , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Osteoblastos/citologia , Osteoporose Pós-Menopausa/terapia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
12.
J Med Chem ; 64(13): 9078-9099, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34129329

RESUMO

Fibroblast growth factor receptors (FGFRs) have become promising therapeutic targets in various types of cancers. In fact, several selective irreversible inhibitors capable of covalently reacting with the conserved cysteine of FGFRs are currently being evaluated in clinical trials. In this article, we optimized and discovered a novel lead compound 36 with remarkable inhibitory effects against FGFR (1-3), which is a derivative of 2H-pyrazolo[3,4-d]pyrimidine. The irreversible binding to FGFRs was characterized by LC-MS. This compound has been shown to exhibit significant anti-proliferation effects against NCI-H1581 and SNU-16 cancer cell lines both in vitro and in vivo. Compound 36 has also demonstrated a low toxicity profile and adequate pharmacokinetic properties and is currently under validation as a potential drug candidate.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
13.
PLoS Biol ; 19(6): e3001311, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34181639

RESUMO

Proteins of the major histocompatibility complex class I (MHC I), predominantly known for antigen presentation in the immune system, have recently been shown to be necessary for developmental neural refinement and adult synaptic plasticity. However, their roles in nonneuronal cell populations in the brain remain largely unexplored. Here, we identify classical MHC I molecule H2-Kb as a negative regulator of proliferation in neural stem and progenitor cells (NSPCs). Using genetic knockout mouse models and in vivo viral-mediated RNA interference (RNAi) and overexpression, we delineate a role for H2-Kb in negatively regulating NSPC proliferation and adult hippocampal neurogenesis. Transcriptomic analysis of H2-Kb knockout NSPCs, in combination with in vitro RNAi, overexpression, and pharmacological approaches, further revealed that H2-Kb inhibits cell proliferation by dampening signaling pathways downstream of fibroblast growth factor receptor 1 (Fgfr1). These findings identify H2-Kb as a critical regulator of cell proliferation through the modulation of growth factor signaling.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Envelhecimento/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Hipocampo/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese
14.
Cells ; 10(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068816

RESUMO

Lung cancer is the most common cause of cancer-related deaths globally. Genetic alterations, such as amplifications, mutations and translocations in the fibroblast growth factor receptor (FGFR) family have been found in non-small cell lung cancer (NSCLC) where they have a role in cancer initiation and progression. FGFR aberrations have also been identified as key compensatory bypass mechanisms of resistance to targeted therapy against mutant epidermal growth factor receptor (EGFR) and mutant Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) in lung cancer. Targeting FGFR is, therefore, of clinical relevance for this cancer type, and several selective and nonselective FGFR inhibitors have been developed in recent years. Despite promising preclinical data, clinical trials have largely shown low efficacy of these agents in lung cancer patients with FGFR alterations. Preclinical studies have highlighted the emergence of multiple intrinsic and acquired resistance mechanisms to FGFR tyrosine kinase inhibitors, which include on-target FGFR gatekeeper mutations and activation of bypass signalling pathways and alternative receptor tyrosine kinases. Here, we review the landscape of FGFR aberrations in lung cancer and the array of targeted therapies under clinical evaluation. We also discuss the current understanding of the mechanisms of resistance to FGFR-targeting compounds and therapeutic strategies to circumvent resistance. Finally, we highlight our perspectives on the development of new biomarkers for stratification and prediction of FGFR inhibitor response to enable personalisation of treatment in patients with lung cancer.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Anticorpos Monoclonais , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular , Cromossomos , Ensaios Clínicos como Assunto , Receptores ErbB/metabolismo , Genes ras , Humanos , Mutação , Mutação Puntual , Translocação Genética
15.
Cells ; 10(6)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070622

RESUMO

Fibroblast growth factor (FGF) signaling is involved in the pathogenesis of multiple sclerosis (MS). Data from neuropathology studies suggest that FGF signaling contributes to the failure of remyelination in MS. In MOG35-55-induced EAE, oligodendrocyte-specific deletion of FGFR1 and FGFR2 resulted in a less severe disease course, reduced inflammation, myelin and axon degeneration and changed FGF/FGFR and BDNF/TrkB signaling. Since signaling cascades in oligodendrocytes could not be investigated in the EAE studies, we here aimed to characterize FGFR-dependent oligodendrocyte-specific signaling in vitro. FGFR inhibition was achieved by application of the multi-kinase-inhibitor dovitinib and the FGFR1/2/3-inhibitor AZD4547. Both substances are potent inhibitors of FGF signaling; they are effective in experimental tumor models and patients with malignancies. Effects of FGFR inhibition in oligodendrocytes were studied by immunofluorescence microscopy, protein and gene analyses. Application of the tyrosine kinase inhibitors reduced FGFR1, phosphorylated ERK and Akt expression, and it enhanced BDNF and TrkB expression. Furthermore, the myelin proteins CNPase and PLP were upregulated by FGFR inhibition. In summary, inhibition of FGFR signaling in oligodendrocytes can be achieved by application of tyrosine kinase inhibitors. Decreased phosphorylation of ERK and Akt is associated with an upregulation of BDNF/TrkB signaling, which may be responsible for the increased production of myelin proteins. Furthermore, these data suggest that application of FGFR inhibitors may have the potential to promote remyelination in the CNS.


Assuntos
Benzamidas/farmacologia , Benzimidazóis/farmacologia , Esclerose Múltipla/tratamento farmacológico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Quinolonas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Remielinização/efeitos dos fármacos , Animais , Linhagem Celular , Oligodendroglia , Ratos
16.
Cells ; 10(5)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068954

RESUMO

Increasing evidence indicates that success of targeted therapies in the treatment of cancer is context-dependent and is influenced by a complex crosstalk between signaling pathways and between cell types in the tumor. The Fibroblast Growth Factor (FGF)/FGF receptor (FGFR) signaling axis highlights the importance of such context-dependent signaling in cancer. Aberrant FGFR signaling has been characterized in almost all cancer types, most commonly non-small cell lung cancer (NSCLC), breast cancer, glioblastoma, prostate cancer and gastrointestinal cancer. This occurs primarily through amplification and over-expression of FGFR1 and FGFR2 resulting in ligand-independent activation. Mutations and translocations of FGFR1-4 are also identified in cancer. Canonical FGF-FGFR signaling is tightly regulated by ligand-receptor combinations as well as direct interactions with the FGFR coreceptors heparan sulfate proteoglycans (HSPGs) and Klotho. Noncanonical FGFR signaling partners have been implicated in differential regulation of FGFR signaling. FGFR directly interacts with cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins, contributing to invasive and migratory properties of cancer cells, whereas interactions with other receptor tyrosine kinases (RTKs) regulate angiogenic, resistance to therapy, and metastatic potential of cancer cells. The diversity in FGFR signaling partners supports a role for FGFR signaling in cancer, independent of genetic aberration.


Assuntos
Carcinogênese , Neoplasias/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Humanos
17.
PLoS One ; 16(5): e0251202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33989301

RESUMO

Fibroblast growth factor receptor 1 (FGFR1) has recently been identified as a promising novel therapeutic target and prognostic marker in different types of cancer. In the present study, a meta-analysis was performed to clarify the correlation between FGFR1 and the survival outcomes of head and neck squamous cell carcinoma (HNSCC) patients. PubMed, Embase, and Web of Science were systematically searched for relevant studies in order to explore the prognostic significance of FGFR1 in HNSCC. Hazards ratios (HR) and 95% confidence intervals (CI) were collected to estimate the correlation between overexpression and amplification of FGFR1 and survival outcomes of HNSCC patients. Nine studies including 2708 patients with HNSCC were finally selected for the meta-analysis. The results indicated that FGFR1 predicted poor overall survival (OS) (HR, 1.97; 95% CI, 1.49-2.61, P<0.001) in HNSCC patients. Futhermore, FGFR1 was related to poor OS in human papillomavirus (HPV) negative HNSCC not in HPV positive HNSCC patients. Subgroup analysis stratified by molecular abnormalities, such as overexpression or amplification showed the similar results. The present study demonstrated that HNSCC patients with FGFR1 overexpression and amplification were more likely to exhibit poorer survival.


Assuntos
Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Amplificação de Genes/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Pessoa de Meia-Idade , Prognóstico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Adulto Jovem
18.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33999995

RESUMO

The focal adhesion protein Kindlin2 is essential for integrin activation, a process that is fundamental to cell-extracellular matrix adhesion. Kindlin 2 (Fermt2) is widely expressed in mouse embryos, and its absence causes lethality at the peri-implantation stage due to the failure to trigger integrin activation. The function of kindlin2 during embryogenesis has not yet been fully elucidated as a result of this early embryonic lethality. Here, we showed that kindlin2 is essential for neural crest (NC) formation in Xenopus embryos. Loss-of-function assays performed with kindlin2-specific morpholino antisense oligos (MOs) or with CRISPR/Cas9 techniques in Xenopus embryos severely inhibit the specification of the NC. Moreover, integrin-binding-deficient mutants of Kindlin2 rescued the phenotype caused by loss of kindlin2, suggesting that the function of kindlin2 during NC specification is independent of integrins. Mechanistically, we found that Kindlin2 regulates the fibroblast growth factor (FGF) pathway, and promotes the stability of FGF receptor 1. Our study reveals a novel function of Kindlin2 in regulating the FGF signaling pathway and provides mechanistic insights into the function of Kindlin2 during NC specification.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Crista Neural/embriologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Proteínas de Membrana/genética , Morfolinos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Proteínas de Xenopus/genética
19.
Eur J Cancer ; 151: 136-149, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33984662

RESUMO

Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response. In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123. A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123. The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metilação de DNA , Epigênese Genética , Neoplasias Pulmonares/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Biomolecules ; 11(4)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916323

RESUMO

Triple negative breast cancer (TNBC) is associated with unfavorable prognosis and high relapse rates following chemotherapy. There is an urgent need to develop effective targeted therapy for this BC subtype. The type I insulin-like growth factor receptor (IGF-IR) was identified as a potential target for BC management. We previously reported on the production of the IGF-Trap, a soluble IGF-1R fusion protein that reduces the bioavailability of circulating IGF-1 and IGF-2 to the cognate receptor, impeding signaling. In nude mice xenotransplanted with the human TNBC MDA-MB-231 cells, we found variable responses to this inhibitor. We used this model to investigate potential resistance mechanisms to IGF-targeted therapy. We show here that prolonged exposure of MDA-MB-231 cells to the IGF-Trap in vitro selected a resistant subpopulation that proliferated unhindered in the presence of the IGF-Trap. We identified in these cells increased fibroblast growth factor receptor 1 (FGFR1) activation levels that sensitized them to the FGFR1-specific tyrosine kinase inhibitor PD166866. Treatment with this inhibitor caused cell cycle arrest in both the parental and resistant cells, markedly increasing cell death in the latter. When combined with the IGF-Trap, an increase in cell cycle arrest was observed in the resistant cells. Moreover, FGFR1 silencing increased the sensitivity of these cells to IGF-Trap treatment in vivo. Our data identify increased FGFR1 signaling as a resistance mechanism to targeted inhibition of the IGF-IR and suggest that dual IGF-1R/FGFR1 blockade may be required to overcome TNBC cell resistance to IGF-axis inhibitors.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptor IGF Tipo 1/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Ligantes , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/metabolismo
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