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1.
PLoS Pathog ; 16(8): e1008823, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845931

RESUMO

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.


Assuntos
Vírus da Influenza A/metabolismo , Pulmão , Macrófagos , Infecções por Orthomyxoviridae , Proteínas PrPC/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais Murinos/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Mutantes , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Proteínas PrPC/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
2.
PLoS One ; 15(7): e0235850, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673341

RESUMO

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.


Assuntos
Autorrenovação Celular , Glucose/metabolismo , Células-Tronco Neoplásicas/metabolismo , Quinases da Família src/metabolismo , Aldeído Desidrogenase 1/genética , Aldeído Desidrogenase 1/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/fisiologia , Proteoma/genética , Proteoma/metabolismo , Quinases da Família src/genética
3.
Proc Natl Acad Sci U S A ; 117(27): 15809-15817, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571924

RESUMO

Src family kinase Lck plays critical roles during T cell development and activation, as it phosphorylates the TCR/CD3 complex to initiate TCR signaling. Lck is present either in coreceptor-bound or coreceptor-unbound (free) forms, and we here present evidence that the two pools of Lck have different molecular properties. We discovered that the free Lck fraction exhibited higher mobility than CD8α-bound Lck in OT-I T hybridoma cells. The free Lck pool showed more activating Y394 phosphorylation than the coreceptor-bound Lck pool. Consistent with this, free Lck also had higher kinase activity, and free Lck mediated higher T cell activation as compared to coreceptor-bound Lck. Furthermore, the coreceptor-Lck coupling was independent of TCR activation. These findings give insights into the initiation of TCR signaling, suggesting that changes in coreceptor-Lck coupling constitute a mechanism for regulation of T cell sensitivity.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/metabolismo , Quinases da Família src/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Hibridomas/imunologia , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Camundongos , Fosforilação/genética , Ligação Proteica/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/imunologia
4.
PLoS One ; 15(4): e0231739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352989

RESUMO

OBJECTIVES: We previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvß3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation. METHODS AND RESULTS: Using intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition. CONCLUSION: The FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.


Assuntos
Permeabilidade Capilar/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Hemorragia/patologia , Microvasos/patologia , Quinases da Família src/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Fibrinogênio/toxicidade , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Hemorragia/induzido quimicamente , Humanos , Microscopia Intravital , Pulmão/irrigação sanguínea , Masculino , Mesentério/irrigação sanguínea , Mesentério/diagnóstico por imagem , Microvasos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/genética
5.
Gene ; 747: 144700, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32330537

RESUMO

OBJECTIVE: Survival rate of laryngeal squamous cell carcinoma (LSCC) patients is not improving. To understand more complete biology of LSCC, studies focused on identification of new specific and prognostic markers are performed. The aim of current study was to evaluate the impact of five different single nucleotide polymorphisms (SNP) (IL6 rs1800795, BLK rs13277113, TIMP3 rs9621532, IL1RL1 rs1041973 and IL1RAP rs4624606) on LSCC development. MATERIAL AND METHODS: A total of 891 subjects (353 histologically verified LSCC patients and 538 healthy controls) were involved in this study. The genotyping was carried out using the real-time-PCR. RESULTS: Statistical analysis revealed statistically significant associations between TIMP3 rs96215332 variants and LSCC in the codominant (OR = 0.600; 95% CI: 0.390-0.922; p = 0.020), overdominant (OR = 0.599; 95% CI: 0.390-0.922; p = 0.020) and additive (OR = 0.675; 95% CI: 0.459-0.991; p = 0.045) models. Also, significant variants of IL1RAP rs4624606 were determined in the codominant (OR = 1.372; 95% CI: 1.031-1.827; p = 0.030), overdominant (OR = 1.353; 95% CI: 1.018-1.798; p = 0.037) and additive (OR = 1.337; 95% CI: 1.038-1.724; p = 0.025) models. CONCLUSION: Results of the current study indicate significant associations between TIMP3 rs9621532 and IL1RAP rs4624606 gene polymorphisms and LSCC development.


Assuntos
Carcinoma de Células Escamosas/genética , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-6/genética , Neoplasias Laríngeas/genética , Polimorfismo de Nucleotídeo Único/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Quinases da Família src/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas/genética
6.
Anticancer Res ; 40(4): 1989-1996, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234888

RESUMO

BACKGROUND/AIM: The antitumor effect of sustained calcium supply on Src degradation was investigated in the context of hormone-dependent breast cancer, followed by elucidation of the underlying mechanisms. MATERIALS AND METHODS: Hormone-dependent T-47D breast cancer cells were used. Lactate calcium salt (LCS) was used as the source of sustained calcium supply, and the applicable concentration of LCS was determined by the colorimetric MTT assay. LCS-mediated deactivation of downstream signaling via Src degradation was identified by western blot and immunocytochemistry. RESULTS: Calcium-mediated degradation of Src decreased survival signaling via phosphoinositide 3-kinase and protein kinase B and resulted in significant inhibition of the clonogenic ability of hormone-dependent breast cancer cells. Tumor volume was significantly decreased in response to LCS injection in a heterotopic xenograft model, and immuno histochemistry revealed tumor necrosis. CONCLUSION: Sustained supply of calcium inhibited survival signaling via degradation of Src in hormone-dependent breast cancer cells.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Proteólise/efeitos dos fármacos , Quinases da Família src/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cálcio/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Ácido Láctico/farmacologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores
7.
BMC Med Genet ; 21(1): 24, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32028929

RESUMO

BACKGROUND: Maturity-onset diabetes of the young (MODY) is a genetically and clinically heterogeneous group of hereditary diabetes, generally caused by one abnormal gene. MODY5 is caused by mutations of the hepatocyte nuclear factor 1 homeobox ß gene (HNF1ß), always as a part of Chr17q12 deletion, whereas heterozygous mutation in B lymphocyte kinase (BLK) gene is responsible for MODY11. CASE PRESENTATION: We report a patient who developed diabetes with a 1.58-Mb Chr17q12 microdeletion and BLK gene c.211G > A mutation using the cytoscan high-density array and whole-exome sequencing analysis. The patient received the surgery at five days after birth for the duodenal atresia and had normal growth postoperatively. Mild elevated liver enzymes were found along with the normal renal function. Quantitative analysis of ß-cell function markers, including fasting insulin (< 0.2 mIU/L), fasting C-peptide (0.02 µg/L), postprandial-2 h insulin (< 0.2 mIU/L), and postprandial-2 h C-peptide (0.03 µg/L) suggested a severe loss of insulin secreting capacity. Meanwhile, islet autoantibodies (GADA, IA-2, ICA, and IAA) in the patient's blood appeared negative. Neither dysplasia in other tissues nor abnormality in development and behavior was found. CONCLUSION: To date, gastrointestinal malformations were extremely rarely reported in patients with MODY. Our clinical report further expands the clinical presentation and variability of MODY5.


Assuntos
Diabetes Mellitus Tipo 2/genética , Obstrução Duodenal/genética , Fator 1-beta Nuclear de Hepatócito/genética , Atresia Intestinal/genética , Quinases da Família src/genética , Diabetes Mellitus Tipo 2/patologia , Obstrução Duodenal/patologia , Feminino , Humanos , Recém-Nascido , Insulina/genética , Atresia Intestinal/patologia , Masculino , Mutação/genética , Fenótipo
8.
FASEB J ; 34(1): 66-81, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914639

RESUMO

Angiogenesis is critical for the development, progression, and metastasis of hepatocellular carcinoma (HCC), but the roles of miR-3064-5p in HCC angiogenesis are still unknown. In this study, the roles of miR-3064-5p in HCC angiogenesis were studied in 192 HCC patients, xenograft mouse models, and HCC cell lines. The results showed that miR-3064-5p expression was significantly decreased in HCC tissues and cells, and downregulated miR-3064-5p was associated with upregulated angiogenic potential of HCC. MiR-3064-5p inhibited proangiogenic VEGFA and angiogenin expressions but induced antiangiogenic endostatin and MMP12 expressions, finally leading to suppression of HCC angiogenesis, as shown by the decline in intratumoral microvessel density (MVD). Moreover, miR-3064-5p was inversely correlated with lncRNA MALAT1 and FOXA1. FOXA1 bound to and interacted with CD24 and then regulated Src phosphorylation. MiR-3064-5p played an antiangiogenic role by inhibiting the FOXA1/CD24/Src pathway, whereas oncogenic MALAT1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-3064-5p to alleviate the suppressive effect on the FOXA1 pathway. HCC patients with high miR-3064-5p, low MALAT1, or low FOXA1 expression had a better prognosis with longer overall survival and recurrence-free survival. In univariate and multivariate analyses, miR-3064-5p was identified as the independent prognostic predicator for HCC progression and patient survival. Taken together, miR-3064-5p exerts an antiangiogenic role by targeting the FOXA1/CD24/Src pathway but oncogenic lncRNA MALAT1 acts as a ceRNA to sponge miR-3064-5p. MiR-3064-5p is of great clinical significance and is a novel prognostic indicator and an attractive therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/irrigação sanguínea , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/irrigação sanguínea , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Antígeno CD24/genética , Antígeno CD24/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Neoplasias Hepáticas/patologia , Camundongos , MicroRNAs/genética , Neoplasias Experimentais , RNA Longo não Codificante/genética , Quinases da Família src/genética , Quinases da Família src/metabolismo
9.
PLoS One ; 15(1): e0227855, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999741

RESUMO

The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Carcinoma/genética , Neoplasias da Próstata/genética , Quinases da Família src/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Cortactina/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatina/genética , Gelatina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação/genética , Fosforilação , Podossomos/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios de Homologia de src/genética
10.
J Biomed Sci ; 27(1): 2, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31898491

RESUMO

BACKGROUND: Serglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined. METHODS: Overexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining. RESULTS: SRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation. CONCLUSIONS: Proteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Glicosaminoglicanos/genética , Receptores de Hialuronatos/genética , Proteoglicanas/genética , Proteínas de Transporte Vesicular/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Glicosaminoglicanos/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Proteoglicanas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases da Família src/genética
11.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 49-57, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31828293

RESUMO

Gastric cancer (GC) is one of malignant tumors with high mortality and morbidity in the world. MicroRNA-122 (miR-122) acts as a tumor suppressor in a variety of cancers and has been found to be dominant in gastric adenocarcinoma. However, the specific biological function of miR-122-5p in GC is not completely clear. In this study, we found that miR-122-5p was low-expressed in GC tissues and cell lines by using qRT-PCR. Overexpression of miR-122-5p inhibited the proliferation, migration, and invasion of GC cells by using CCK-8 and transwell assays. On the contrary, downregulation of miR-122-5p promoted the proliferation, migration, and invasion of GC cells. In addition, we found that the expression of LYN, an Src family tyrosine kinase, was inversely correlated with miR-122-5p expression in GC tissues by using western blot analysis, immunohistochemistry, and qRT-PCR assays. Meanwhile, luciferase assay results indicated that LYN is a direct target of miR-122-5p in GC cells. Moreover, silencing LYN expression by its siRNA inhibited the proliferation, migration, and invasion of GC cells. Importantly, overexpression of LYN restored miR-122-5p-mediated inhibition of the proliferation, migration, and invasion of GC cells. Taken together, our results indicated miR-122-5p inhibited the proliferation, migration, and invasion by targeting LYN in GC.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Neoplasias Gástricas/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mimetismo Molecular/genética , Mutação , RNA Interferente Pequeno/genética , Neoplasias Gástricas/patologia , Transfecção
12.
Oncogene ; 39(2): 308-321, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31477830

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human cancers, with 5-year patient survival rates of <5%. Activating mutations in KRAS are the predominant oncogenic drivers of PDAC but are accompanied by additional lower frequency genetic alterations. Our group previously identified the guanine nucleotide exchange factor ARHGEF10 in a genomic screen for genes with copy number alterations that may synergize with oncogenic KRAS to promote PDAC carcinogenesis. In the present study we show that ARHGEF10 possesses putative tumor suppressor function in PDAC. ARHGEF10 expression is reduced in over 70% of PDAC cell lines, and copy number loss is documented in more than 30% of PDAC patient-derived xenografts. Loss of ARHGEF10 expression enhanced subcutaneous tumor growth in mouse models, while its exogenous expression greatly impaired tumorigenesis. Loss of ARHGEF10 expression also increased in vitro proliferation, invasion, and motility of PDAC cell lines, and enhanced their metastatic spread in orthotopic mouse models. Treatment of ARHGEF10-depleted cells with the inhibitor dasatinib reduced levels of phospho Src kinase and attenuated motility and invasion in vitro. Together, our data indicate that ARHGEF10 may function as a tumor suppressor in PDAC.


Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Quinases da Família src/genética , Adenocarcinoma/patologia , Animais , Carcinogênese/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dasatinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética
13.
Immunology ; 159(3): 279-288, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31670388

RESUMO

Genome-wide association studies (GWAS) have identified many loci for systemic lupus erythematosus (SLE). However, identification of functionally relevant genes remains a challenge. The aim of this study was to highlight potential causal genes for SLE in the GWAS loci. By applying Mendelian randomization (MR) methods, such as summary data-based MR (SMR), generalized SMR and MR pleiotropy residual sum and outlier, we identified DNA methylations in 15 loci and mRNA expression of 21 genes that were causally associated with SLE. The identified genes enriched in 14 specific KEGG pathways (e.g. SLE, viral carcinogenesis) and two GO terms (interferon-γ-mediated signaling pathway and innate immune response). Among the identified genes, UBE2L3 and BLK variants were significantly associated with UBE2L3 and BLK methylations and gene expressions, respectively. UBE2L3 was up-regulated in SLE patients in several types of immune cells. Methylations (e.g. cg06850285) and mRNA expression of UBE2L3 were causally associated with SLE. Methylation site cg09528494 and mRNA expression of BLK were causally associated with SLE. BLK single nucleotide polymorphisms that were significantly associated with SLE were strongly associated with plasma cathepsin B level. Deep analysis identified that plasma cathepsin B level was causally associated with SLE. In summary, this study identified hundreds of DNA methylations and genes as potential risk factors for SLE. Genetic variants in UBE2L3 gene might affect SLE by influencing gene expression. Genetic variants in BLK gene might affect SLE by influencing BLK gene expression and plasma cathepsin B protein level.


Assuntos
Metilação de DNA , Epigênese Genética , Lúpus Eritematoso Sistêmico/genética , Enzimas de Conjugação de Ubiquitina/genética , Quinases da Família src/genética , Catepsina B/sangue , Bases de Dados Genéticas , Marcadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Análise da Randomização Mendeliana , Fenótipo , Polimorfismo de Nucleotídeo Único , Fatores de Risco
14.
Biochim Biophys Acta Gen Subj ; 1864(1): 129457, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678144

RESUMO

BACKGROUND: Adenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR. METHODS: We investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation. RESULTS: A2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression. CONCLUSION: Activation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage. GENERAL SIGNIFICANCE: Our results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.


Assuntos
Neoplasias Pulmonares/radioterapia , Tolerância a Radiação/genética , Receptor A2B de Adenosina/genética , Células A549 , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Histonas/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosforilação/efeitos da radiação , Radiação , Radiossensibilizantes/farmacologia , Translocação Genética/efeitos dos fármacos , Translocação Genética/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Quinases da Família src/genética
15.
PLoS One ; 14(12): e0225887, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790499

RESUMO

Unregulated protein-tyrosine kinase signaling is a common feature of AML, often involving mutations in Flt3 and overexpression of myeloid Src-family kinases (Hck, Fgr, Lyn). Here we show that high-level expression of these Src kinases predicts poor survival in a large cohort of AML patients. To test the therapeutic benefit of Flt3 and Src-family kinase inhibition, we used the pyrrolopyrimidine kinase inhibitor A-419259. This compound potently inhibits Hck, Fgr, and Lyn as well as Flt3 bearing an activating internal tandem duplication (ITD). Flt3-ITD expression sensitized human TF-1 myeloid cells to growth arrest by A-419259, supporting direct action on the Flt3-ITD kinase domain. Cells transformed with the Flt3-ITD mutants D835Y and F691L were resistant to A-419259, while co-expression of Hck or Fgr restored inhibitor sensitivity to Flt3-ITD D835Y. Conversely, Hck and Fgr mutants with engineered A-419259 resistance mutations decreased sensitivity of TF-1/Flt3-ITD cells. To investigate de novo resistance mechanisms, A-419259-resistant Flt3-ITD+ AML cell populations were derived via long-term dose escalation. Whole exome sequencing identified a distinct Flt3-ITD kinase domain mutation (N676S/T) among all A-419259 target kinases in each of six independent resistant cell populations. These studies show that Hck and Fgr expression influences inhibitor sensitivity and the pathway to acquired resistance in Flt3-ITD+ AML.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda , Mutação de Sentido Incorreto , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-hck , Proteínas Proto-Oncogênicas , Pirimidinas/farmacologia , Pirróis/farmacologia , Tirosina Quinase 3 Semelhante a fms , Quinases da Família src , Substituição de Aminoácidos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Prognóstico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck/biossíntese , Proteínas Proto-Oncogênicas c-hck/genética , Sequenciamento Completo do Exoma , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Quinases da Família src/biossíntese , Quinases da Família src/genética
16.
Int J Mol Sci ; 20(24)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847183

RESUMO

Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3's ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylation. Using native and bottom-up mass spectrometry, we performed detailed mapping and characterization of human EYA3 Src-phosphorylation sites. Thirteen tyrosine residues with different phosphorylation and autodephosphorylation kinetics were detected. Among these, Y77, 96, 237, and 508 displayed an increased resistance to autodephosphorylation. Y77 and Y96 were found to have the highest impact on the overall EYA3 phosphorylation. Using cell cycle analysis, we showed that Y77, Y96, and Y237 are involved in HEK293T proliferation. Mutation of the three tyrosine residues abolished the pro-proliferative effect of EYA3 overexpression. We have also identified a Src-induced phosphorylation pattern of EYA3 in these cells. These findings suggest that EYA3's tyrosine phosphorylation sites are non-equivalent with their phosphorylation levels being under the control of Src-kinase activity and of EYA3's autodephosphorylation.


Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Quinases da Família src/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Fosforilação , Proteínas Tirosina Fosfatases/genética , Tirosina/genética , Tirosina/metabolismo , Quinases da Família src/genética
17.
Mol Cells ; 42(11): 810-819, 2019 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-31707778

RESUMO

For physiological or pathological understanding of bone disease caused by abnormal behavior of osteoclasts (OCs), functional studies of molecules that regulate the generation and action of OCs are required. In a microarray approach, we found the suppression of tumorigenicity 5 (ST5) gene is upregulated by receptor activator of nuclear factor-κB ligand (RANKL), the OC differentiation factor. Although the roles of ST5 in cancer and ß-cells have been reported, the function of ST5 in bone cells has not yet been investigated. Knockdown of ST5 by siRNA reduced OC differentiation from primary precursors. Moreover, ST5 downregulation decreased expression of NFATc1, a key transcription factor for osteoclastogenesis. In contrast, overexpression of ST5 resulted in the opposite phenotype of ST5 knockdown. In immunocytochemistry experiments, the ST5 protein is colocalized with Src in RANKL-committed cells. In addition, ST5 enhanced activation of Src and Syk, a Src substrate, in response to RANKL. ST5 reduction caused a decrease in RANKL-evoked calcium oscillation and inhibited translocation of NFATc1 into the nucleus. Taken together, these findings provide the first evidence of ST5 involvement in positive regulation of osteoclastogenesis via Src/Syk/calcium signaling.


Assuntos
Sinalização do Cálcio/genética , Proteínas de Ligação a DNA/genética , Osteoclastos/metabolismo , Osteogênese/genética , Quinase Syk/genética , Proteínas Supressoras de Tumor/genética , Quinases da Família src/genética , Animais , Reabsorção Óssea/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos ICR , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Interferência de RNA , Quinase Syk/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Quinases da Família src/metabolismo
18.
Int J Mol Sci ; 20(21)2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31690042

RESUMO

Receptor tyrosine kinases (RTKs) play important roles in the pathogenic processes of kidney fibrosis. However, the pathophysiological roles of recepteur d'origine nantais (RON), one of the receptor tyrosine kinases, have not yet been defined. We investigated whether the activation or sequence-specific small interfering RNA (siRNA) suppression of RON could regulate epithelial mesenchymal transition (EMT) and the expression of pro-fibrotic markers, and its underlying molecular mechanisms. Stable cell lines and transient transfection for RON and the transfected cells of siRNA for RON were developed to investigate the molecular mechanisms in human kidney proximal tubular epithelial (HK-2) and interstitial fibroblasts (NRK49F) cells. RON overexpression induced EMT and increased expression of fibrosis-related proteins such as N-cadherin, vimentin, transforming growth factor-ß (TGFß), αSMA, and fibronectin in HK-2 and NRK49F cells. RON overexpression increased various RTKs and the phosphorylation of Src (Y416) and Smad, while inhibition of RON by siRNA attenuated the expression of EMT- and fibrosis-related proteins and decreased RTKs such as insulin-like growth factor receptor (IGFR), fibroblast growth factor receptor 1 (FGFR1), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR), as well as the phosphorylation of Src and Smad pathways. siRNA silencing of Src also attenuated the expression of IGFR, FGFR1, VEGFR, and PDGFR. Inhibition of RON can exert an anti-fibrotic effect by the inhibition of EMT and other RTKs through control of Src and Smad pathways in HK-2 and NRK49F cells.


Assuntos
Transição Epitelial-Mesenquimal , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Quinases da Família src/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Smad/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Quinases da Família src/genética
19.
J Cell Biol ; 218(9): 3077-3097, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31420453

RESUMO

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0-2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.


Assuntos
Microtúbulos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Animais , Técnicas Biossensoriais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Microtúbulos/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteína rhoA de Ligação ao GTP/genética , Quinases da Família src/genética
20.
Mol Syst Biol ; 15(8): e8849, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31464373

RESUMO

Obesity-associated type 2 diabetes and accompanying diseases have developed into a leading human health risk across industrialized and developing countries. The complex molecular underpinnings of how lipid overload and lipid metabolites lead to the deregulation of metabolic processes are incompletely understood. We assessed hepatic post-translational alterations in response to treatment of cells with saturated and unsaturated free fatty acids and the consumption of a high-fat diet by mice. These data revealed widespread tyrosine phosphorylation changes affecting a large number of enzymes involved in metabolic processes as well as canonical receptor-mediated signal transduction networks. Targeting two of the most prominently affected molecular features in our data, SRC-family kinase activity and elevated reactive oxygen species, significantly abrogated the effects of saturated fat exposure in vitro and high-fat diet in vivo. In summary, we present a comprehensive view of diet-induced alterations of tyrosine signaling networks, including proteins involved in fundamental metabolic pathways.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Fosfotirosina/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Ácidos Graxos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/genética , Obesidade/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Ratos , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Quinases da Família src/genética , Quinases da Família src/metabolismo
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