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1.
EBioMedicine ; 49: 55-71, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31685442

RESUMO

BACKGROUND: Although bone morphogenetic protein 6 (BMP6) signaling pathway has been implicated in many types of cancer, its role of tumorigenesis seems to be controversial and its ubiquitin-modifying mechanisms have not been fully addressed. Our study was designed to investigate how BMP6 signaling pathway is regulated by ubiquitin-modifying systems and to address molecular and clinical significance in colorectal cancers. METHODS: Human deubiquitnase (DUB) siRNA library was used to screen the specific DUB, named PSMD14, involved in BMP6 signaling pathway. Immunoblot, immunoprecipitation and ubiquitination assays were used to analyze targets of the PSMD14. A role of PSMD14-mediated BMP6 signaling pathway for malignant cancer progression was investigated using in vitro and in vivo model of colorectal cancers as well as clinical samples of colorectal cancer patients. FINDINGS: The deubiquitinase PSMD14 acts as a positive regulator for the initiation of the BMP6 signaling pathway through deubiquitinating K48-linked ALK2 type I receptor ubiquitination mediated by Smurf1 E3 ligase, resulting in increased stability of the ALK2. This role of PSMD14 is independent of its intrinsic role in the 26S proteasome system. Furthermore, either PSMD14 or ALK2 depletion significantly decreases tumorigenesis of HCT116 colorectal cancer cells in a xenograft model as well as cancer stemness/chemoresistance, and expression of the PSMD14 and ALK2 gene are correlated with malignant progression and the survival of colorectal cancer patients. INTERPRETATION: These findings suggest that the PSMD14-ALK2 axis plays an essential role in initiation of the BMP6 signaling pathway and contributes to tumorigenesis and chemoresistance of colorectal cancers.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Lisina/metabolismo , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Poliubiquitina/metabolismo , Prognóstico , Ligação Proteica , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
Sci Adv ; 5(8): eaav6789, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31489365

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Células Endoteliais/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Biomimética/métodos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Linhagem Celular Tumoral , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/patologia
3.
Cancer Sci ; 110(11): 3486-3496, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31483918

RESUMO

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation- and apoptosis-inducing effects on glioma-initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin-producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP-2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP-2-induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK-2, which was associated with BMP-induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain-of-function of EPHA6 together with BMP-2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP-2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Apoptose , Proteína Morfogenética Óssea 2/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Receptor EphA6/metabolismo , Animais , Proteína Morfogenética Óssea 2/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Prognóstico , Proteínas Supressoras de Tumor/metabolismo
4.
Mol Med Rep ; 20(4): 2979-2989, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432174

RESUMO

Heterotopic ossification (HO) refers to the appearance of osteoblasts in soft tissues under pathological conditions, such as trauma or infection. HO arises in an unpredictable way without any recognizable initiation. Activin receptor­like kinase­2 (ALK2) is a type I cell surface receptor for bone morphogenetic proteins (BMPs). The dysregulation of ALK2 signaling is associated with a variety of diseases, including cancer and HO. At present, the prevention and treatment of HO in the clinic predominantly includes nonsteroidal anti­inflammatory drugs (NSAIDs), bisphosphonates and other drug treatments, low­dose local radiation therapy and surgical resection, rehabilitation treatment and physical therapy. However, most of these therapies have adverse effects. These methods do not prevent the occurrence of HO. The pathogenesis of HO is not being specifically targeted; the current treatment strategies target the symptoms, not the disease. These treatments also cannot solve the fundamental problem of the occurrence of HO. Therefore, scholars have been working to develop targeted therapies based on the pathogenesis of HO. The present review focuses on advances in the understanding of the underlying mechanisms of HO, and possible options for the prevention and treatment of HO. In addition, the role of ALK2 in the process of HO is introduced and the progress made towards the targeted inhibition of ALK2 is discussed. The present study aims to offer a platform for further research on possible targets for the prevention and treatment of HO.


Assuntos
Receptores de Ativinas Tipo I , Sistemas de Liberação de Medicamentos , Ossificação Heterotópica , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Humanos , Ossificação Heterotópica/tratamento farmacológico , Ossificação Heterotópica/enzimologia
5.
EMBO Mol Med ; 11(9): e10567, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31373426

RESUMO

Heterotopic ossification (HO) is the pathological formation of ectopic endochondral bone within soft tissues. HO occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). FOP patients carry a conserved mutation in ACVR1 that becomes neomorphic for activin A responses. Here, we demonstrate the efficacy of BYL719, a PI3Kα inhibitor, in preventing HO in mice. We found that PI3Kα inhibitors reduce SMAD, AKT, and mTOR/S6K activities. Inhibition of PI3Kα also impairs skeletogenic responsiveness to BMPs and the acquired response to activin A of the Acvr1R206H allele. Further, the efficacy of PI3Kα inhibitors was evaluated in transgenic mice expressing Acvr1Q207D . Mice treated daily or intermittently with BYL719 did not show ectopic bone or cartilage formation. Furthermore, the intermittent treatment with BYL719 was not associated with any substantial side effects. Therefore, this work provides evidence supporting PI3Kα inhibition as a therapeutic strategy for HO.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Ossificação Heterotópica/enzimologia , Ossificação Heterotópica/prevenção & controle , Inibidores de Fosfoinositídeo-3 Quinase/administração & dosagem , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Ativinas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Camundongos , Ossificação Heterotópica/genética , Tiazóis/administração & dosagem
6.
Cells ; 8(6)2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31174355

RESUMO

Bone morphogenetic protein (BMP) and Notch signaling are critical for endothelial cell (EC) differentiation in vascular development. Recent studies have shown that excess BMP activity induces Notch signaling in cerebral ECs resulting in arteriovenous malformation (AVMs). However, it is unclear how the crosstalk between BMP and Notch signaling affects cerebral EC differentiation at the gene regulatory level. Here, we report that BMP6 activates the activin receptor-like kinase (ALK) 3, a BMP type 1 receptor, to induce Notch1 receptor and Jagged1 and Jagged2 ligands. We show that increased expression of the Notch components alters the transcriptional regulatory complex in the SRY-Box 2 (Sox2) promoter region so as to induce its expression in cerebral ECs. Together, our results identify Sox2 as a direct target of BMP and Notch signaling and provide information on how altered BMP and Notch signaling affects the endothelial transcriptional landscape.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Encéfalo/citologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteína Jagged-2/genética , Proteína Jagged-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch1/genética , Fatores de Transcrição SOXB1/genética
7.
Dev Cell ; 49(3): 409-424.e6, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31063757

RESUMO

Herein, we report that the TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Ativinas/metabolismo , Neoplasias/metabolismo , Animais , Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Feminino , Xenoenxertos , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos SCID , Metástase Neoplásica , Neoplasias/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral
8.
Commun Biol ; 2: 156, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31098401

RESUMO

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.


Assuntos
Receptores de Ativinas Tipo I/genética , Antineoplásicos/farmacologia , Neoplasias do Tronco Encefálico/tratamento farmacológico , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Administração Oral , Animais , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/mortalidade , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Proliferação de Células , Criança , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/mortalidade , Glioma Pontino Intrínseco Difuso/patologia , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Feminino , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos SCID , Mutação , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/farmacocinética , Piridinas/farmacocinética , Pirimidinas/farmacocinética , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cell Commun Signal ; 17(1): 45, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101053

RESUMO

BACKGROUND: Endometriosis, characterized by the presence of functional endometrial tissues outside the uterus, is one of the most common gynecological disorders. Endometrial mesenchymal stem cells (MSCs) are crucial for the occurrence and development of endometriosis. Ectopic endometrial MSCs exist in the peritoneal cavity. Thus, the bioactive factors in endometriotic peritoneal fluid may regulate the biological behaviors of endometrial MSCs. METHODS: In this study, after assessing the concentration of Activin A in peritoneal fluid using ELISA, we isolated and cultured endometrial MSCs and investigated whether Activin A stimulated endometrial MSCs to differentiate into myofibroblasts and clarified the underlying mechanisms by quantitative real-time PCR, Western blot analysis, immunofluorescent staining, RNA interference and Chromatin immunoprecipitation. We also employed the inhibitors of Activin A to explore the possibility of suppressing the development of fibrosis in endometriosis using primary endometrial MSCs cultures and a mouse model of endometriosis. RESULTS: Here, we revealed that Activin A significantly elevated in endometriotic peritoneal fluid and activin receptor-like kinase (ALK4), the specific receptor for Activin A, obviously enhanced in ectopic endometrial MSCs compared with eutopic endometrial MSCs from women with or without endometriosis. Next, we found that Activin A drived myofibroblast differentiation of endometrial MSCs, with extremely enhanced expression of connective tissue growth factor (CTGF). CTGF was shown to be required for Activin A-induced expression of ACTA2, COL1A1 and FN1 in endometrial MSCs. CTGF induction by Activin A in endometrial MSCs involved the activation of Smad2/3, as evidenced by the phosphorylation and nuclear translocation of Smad2/3 as well as the binding of Smad2/3 to CTGF promoter. Furthermore, Smad/CTGF pathway in endometrial MSCs required activation of STAT3 while independent of PI3K, JNK and p-38 pathways. In addition, we also demonstrated that inhibition of Activin A pathway impeded myofibroblast differentiation of endometrial MSCs and ameliorated fibrosis in endometriosis mice. CONCLUSIONS: Activin A promotes myofibroblast differentiation of endometrial mesenchymal stem cells via STAT3-dependent Smad/CTGF pathway. The results provided the first evidence that STAT3 acted as a crucial Activin A downstream mediator to regulate CTGF production. Our data may supplement the stem cell theory of endometriosis and provide the experimental basis to treat endometriosis-associated fibrosis by manipulating Activin A signaling.


Assuntos
Ativinas/metabolismo , Diferenciação Celular , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Endometriose/metabolismo , Miofibroblastos/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Smad/metabolismo , Actinas/genética , Actinas/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Ativinas/genética , Adulto , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Óxidos S-Cíclicos/uso terapêutico , Endometriose/tratamento farmacológico , Endométrio/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Miofibroblastos/citologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-30891432

RESUMO

To grow and cause disease, intracellular pathogens modulate host cell processes. Identifying these processes as well as the mechanisms used by the pathogens to manipulate them is important for the development of more effective therapeutics. As an example, the intracellular parasite Toxoplasma gondii induces a wide variety of changes to its host cell, including altered membrane trafficking, cytoskeletal reorganization, and differential gene expression. Although several parasite molecules and their host targets have been identified that mediate- these changes, few are known to be required for parasite replication. One exception is the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), which is required for parasite replication in an oxygen-dependent manner. Toxoplasma activates HIF-1 by stabilizing the HIF-1α subunit, and this is dependent on the signaling from the Activin-Like Kinase (ALK) receptor superfamily. Here, we demonstrate that specific overexpression of the ALK family member, ALK4, increased HIF-1 activity in Toxoplasma-infected cells, and this increase required ALK4 kinase activity. Moreover, Toxoplasma stimulated ALK4 to dimerize with its co-receptor, ActRII, and also increased ALK4 kinase activity, thereby demonstrating that Toxoplasma activates the ALK4 receptor. ALK4 activation of HIF-1 was independent of canonical SMAD signaling but rather was dependent on the non-canonical Rho GTPase and JNK MAP kinase signaling pathways. Finally, Toxoplasma increased rates of ALK4 ubiquitination and turnover. These data provide the first evidence indicating that ALK4 signaling is a target for a microbial pathogen to manipulate its host cell.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Interações Hospedeiro-Patógeno , Fator 1 Induzível por Hipóxia/biossíntese , Toxoplasma/crescimento & desenvolvimento , Animais , Células Cultivadas , Humanos , Camundongos
11.
J Exp Clin Cancer Res ; 38(1): 107, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819221

RESUMO

BACKGROUND: Pancreatic cancer (PC) represents one of the most aggressive forms of cancer. The role of long non-coding RNAs (lncRNAs) has been highlighted in various malignancies including PC. The aim of the present study was to investigate the effects associated with actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) on the progression of PC and the underlying mechanism. METHODS: Microarray-based gene expression profiling of PC was performed to identify PC-related lncRNAs, after which the expression of AFAP1-AS1 and cancer stem cell (CSC) markers in PC tissues and cells were determined accordingly. The potential microRNA-384 (miR-384) capable of binding to AFAP1-AS1, in addition to its ability to regulate activin receptor A type I (ACVR1) were analyzed. In order to investigate the effect of the AFAP1-AS1/miR-384/ACVR1 axis on self-renewal ability, tumorigenicity, invasion, migration and stemness of PC cells, shRNA-AFAP1-AS1, miR-384 mimic and inhibitor were cloned into cells. RESULTS: High expression of AFAP1-AS1 and ACVR1 with low expression of miR-384 were detected in PC tissues. ACVR1 was determined to be down-regulated when miR-384 was overexpressed, while the inhibition of AFAP1-AS1 decreased its ability to binding competitively to miR-384, resulting in the down-regulation of ACVR1 and enhancing miR-384 expression, ultimately inhibiting the progression of PC. The knockdown of AFAP1-AS1 or overexpression of miR-384 was confirmed to impair PC cell self-renewal ability, tumorigenicity, invasion, migration and stemness. CONCLUSIONS: Taken together, AFAP1-AS1 functions as an endogenous RNA by competitively binding to miR-384 to regulate ACVR1, thus conferring inhibitory effects on PC cell stemness and tumorigenicity.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/metabolismo , Adulto , Idoso , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor Cross-Talk/fisiologia
12.
Nat Commun ; 10(1): 1023, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833574

RESUMO

Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor, with approximately 25% of DIPGs harboring activating ACVR1 mutations that commonly co-associate with H3.1K27M mutations. Here we show that in vitro expression of ACVR1 R206H with and without H3.1K27M upregulates mesenchymal markers and activates Stat3 signaling. In vivo expression of ACVR1 R206H or G328V with H3.1K27M and p53 deletion induces glioma-like lesions but is not sufficient for full gliomagenesis. However, in combination with PDGFA signaling, ACVR1 R206H and H3.1K27M significantly decrease survival and increase tumor incidence. Treatment of ACVR1 R206H mutant DIPGs with exogenous Noggin or the ACVR1 inhibitor LDN212854 significantly prolongs survival, with human ACVR1 mutant DIPG cell lines also being sensitive to LDN212854 treatment. Together, our results demonstrate that ACVR1 R206H and H3.1K27M promote tumor initiation, accelerate gliomagenesis, promote a mesenchymal profile partly due to Stat3 activation, and identify LDN212854 as a promising compound to treat DIPG.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Astrocitoma/metabolismo , Neoplasias do Tronco Encefálico/metabolismo , Genoma Humano/genética , Glioma/metabolismo , Histonas/metabolismo , Receptores de Ativinas Tipo I/genética , Animais , Astrocitoma/tratamento farmacológico , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Proteínas de Transporte/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Histonas/genética , Humanos , Camundongos , Mutação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
13.
Biochimie ; 158: 246-256, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30703478

RESUMO

Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein. In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112-150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize "hot spot" residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope. Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies.


Assuntos
Anticorpos Monoclonais Murinos/química , Anticorpos Antineoplásicos/química , Anticorpos Neutralizantes/química , Citometria de Fluxo , Proteínas Ligadas por GPI , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Neoplasias , Receptores de Ativinas Tipo I/imunologia , Receptores de Ativinas Tipo I/metabolismo , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Domínios Proteicos
14.
Cell Signal ; 55: 109-118, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633987

RESUMO

Bone morphogenetic protein 6 (BMP6) and transforming growth factor-ß1 (TGF-ß1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Furin, a member of the subtilisin-like proprotein convertase family, promotes the activation of diverse functional proteins by cleaving protein precursors in the secretory pathway. The aim of this study was to investigate the effect and underlying molecular mechanisms by which BMP6 regulates the expression of furin to increase TGF-ß1 production. Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. Our results show that BMP6 significantly up-regulated the expression of furin and increased the production of TGF-ß1 in hGL cells. Using dual inhibition approaches (kinase receptor inhibitors and small interfering RNA-targeted knockdown), we demonstrate that both activin receptor-like (ALK)2 and ALK3 are involved in the BMP6-induced up-regulation of furin. Additionally, knockdown of furin abolished BMP6-induced increases in TGF-ß1 production. Moreover, knockdown of endogenous SMAD4 reversed the BMP6-induced increase in furin expression. These results indicate that the ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-ß1 in hGL cells. Our findings provide insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells.


Assuntos
Proteína Morfogenética Óssea 6/fisiologia , Furina/metabolismo , Células da Granulosa/metabolismo , Células Lúteas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Células Cultivadas , Feminino , Células da Granulosa/citologia , Humanos , Células Lúteas/citologia , Proteína Smad4/metabolismo
15.
Stem Cell Reports ; 12(1): 98-111, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30595547

RESUMO

Exogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.


Assuntos
Diferenciação Celular , Células Endoteliais/citologia , Fator 2 de Diferenciação de Crescimento/farmacologia , Linfangiogênese , Células-Tronco Embrionárias Murinas/citologia , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animais , Calcineurina/metabolismo , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Vasos Linfáticos/citologia , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Methods Mol Biol ; 1891: 155-163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30414131

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a rare human skeletal disease caused by constitutively activating mutations in the gene ACVR1, which encodes a type I BMP/TGFß family member receptor. FOP is characterized by progressive heterotopic ossification (HO) of fibrous tissues, including skeletal muscle, tendons, and ligaments, as well as malformation of the big toes, vertebral fusions, and osteochondromas. Surgical interventions in patients often result in enhanced HO, which can exacerbate rather than improve diagnostic outcomes. As a result of these difficulties, a variety of animal models are needed to study human FOP. Here we describe the methods for creating and characterizing zebrafish conditionally expressing Acvr1lQ204D, the first adult zebrafish model for FOP.


Assuntos
Miosite Ossificante/etiologia , Miosite Ossificante/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Resposta ao Choque Térmico , Humanos , Imuno-Histoquímica , Camundongos Transgênicos , Miosite Ossificante/diagnóstico , Fenótipo , Microtomografia por Raio-X , Peixe-Zebra
17.
Oncogene ; 38(12): 2056-2075, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30401983

RESUMO

Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-ß family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Retinoblastoma/patologia , Transdução de Sinais , Proteína Smad2/metabolismo , Receptores de Ativinas Tipo I/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , Proteína Smad2/genética
18.
J Cell Physiol ; 234(7): 10238-10247, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30417373

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.


Assuntos
Ativinas/farmacologia , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Miosite Ossificante/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Receptores de Ativinas Tipo I/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Miosite Ossificante/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Ligamento Periodontal/metabolismo , Ligamento Periodontal/patologia , Fosforilação , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Adulto Jovem
19.
Chem Biol Interact ; 298: 8-14, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30367833

RESUMO

MicroRNAs (miRNAs) are emerging as important regulators in the pathogenesis of pre-eclampsia (PE). Recent evidence has reported that miR-454 plays an important role in regulating cell proliferation and invasion. The decreased proliferation and invasion of trophoblast cells contribute to the pathogenesis of PE. However, whether miR-454 is involved in the regulation of trophoblast cell proliferation and invasion remains unknown. In this study, we aimed to investigate the potential role and underlying mechanism of miR-454 in regulating trophoblast cell proliferation and invasion in vitro. We found that miR-454 expression was significantly decreased in placental tissues from PE patients compared to controls. Transfection of miR-454 mimics promoted the proliferation, reduced the apoptosis, and increased invasion of trophoblast cells, while transfection of miR-454 inhibitor showed opposite effects. Bioinformatics analysis showed that activin receptor-like kinase 7 (ALK7) was a potential target gene of miR-454. Dual-luciferase reporter assay showed miR-454 directly targeted the 3'-untranslated region of AKL7. Further experiments showed that miR-454 negatively regulated ALK7 expression. Interestingly, transfection of miR-454 mimics significantly abrogated the inhibitory effect of Nodal on trophoblast cell proliferation and invasion. Moreover, overexpression of ALK7 markedly reversed the promotion effect of miR-454 on trophoblast cell proliferation and invasion. Overall, our results suggest that miR-454 promotes the proliferation and invasion of trophoblast cells by downregulation of ALK7. Our study suggests that miR-454 may play critical roles in the pathogenesis of PE and serve as a potential therapeutic target for treatment of PE.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , MicroRNAs/metabolismo , Proteína Nodal/metabolismo , Pré-Eclâmpsia/genética , Trofoblastos/patologia , Regiões 3' não Traduzidas , Receptores de Ativinas Tipo I/genética , Linhagem Celular , Proliferação de Células/genética , Feminino , Humanos , Proteína Nodal/genética , Placenta/patologia , Placenta/fisiologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Transdução de Sinais , Trofoblastos/metabolismo
20.
Mol Pharmacol ; 95(2): 222-234, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30459156

RESUMO

The transforming growth factor ß (TGFß) superfamily includes TGFß, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFß superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor ß receptor 1 (TGFBR1) kinase inhibitors designed to target TGFß signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFß-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFß-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFß- and BMP-regulated signaling pathways to disease states.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA , Camundongos , Células NIH 3T3 , Metástase Neoplásica/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo
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