RESUMO
Long non-coding RNAs (lncRNAs) are aberrantly expressed in many disease conditions, including cancer. Accumulating evidence indicates that some lncRNAs may play critical roles in cancer progression and metastasis. Here, we identify a set of lncRNAs that are upregulated in metastatic subpopulations isolated from colon cancer HCT116 cells in vivo and show that one of these lncRNAs, which we name CALIC, is required for the metastatic activity of colon cancer cells. We show that CALIC associates with the RNA-binding protein hnRNP-L and imparts specificity to hnRNP-L-mediated gene expression. Furthermore, we demonstrate that the CALIC/hnRNP-L complex upregulates the tyrosine kinase receptor AXL and that knockdown of CALIC or AXL using shRNA in colon cancer cells attenuates their ability to form metastases in mice. These results suggest that the CALIC/hnRNP-L complex enhances the metastatic potential of colon cancer cells.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Longo não Codificante/genética , Receptores Proteína Tirosina Quinases/genética , Ribonucleoproteínas/genética , Animais , Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Progressão da Doença , Feminino , Células HCT116 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Many long noncoding RNAs (lncRNAs) are reported to be dysregulated in human cancers and play critical roles in tumor development and progression. Furthermore, it has been reported that many lncRNAs regulate gene expression by recruiting chromatin remodeling complexes to specific genomic loci or by controlling transcriptional or posttranscriptional processes. Here we show that an lncRNA termed UPAT [ubiquitin-like plant homeodomain (PHD) and really interesting new gene (RING) finger domain-containing protein 1 (UHRF1) Protein Associated Transcript] is required for the survival and tumorigenicity of colorectal cancer cells. UPAT interacts with and stabilizes the epigenetic factor UHRF1 by interfering with its ß-transducin repeat-containing protein (TrCP)-mediated ubiquitination. Furthermore, we demonstrate that UHRF1 up-regulates Stearoyl-CoA desaturase 1 and Sprouty 4, which are required for the survival of colon tumor cells. Our study provides evidence for an lncRNA that regulates protein ubiquitination and degradation and thereby plays a critical role in the survival and tumorigenicity of tumor cells. Our results suggest that UPAT and UHRF1 may be promising molecular targets for the therapy of colon cancer.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neoplasias do Colo/genética , RNA Longo não Codificante/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/química , Linhagem Celular Tumoral , Epigênese Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Ubiquitina-Proteína Ligases , Ubiquitinação , Regulação para CimaRESUMO
Lineage development is a stepwise process, governed by stage-specific regulatory factors and associated markers. Astrocytes are one of the principle cell types in the CNS and the stages associated with their development remain very poorly defined. To identify these stages, we performed gene-expression profiling on astrocyte precursor populations in the spinal cord, identifying distinct patterns of gene induction during their development that are strongly correlated with human astrocytes. Validation studies identified a new cohort of astrocyte-associated genes during development and demonstrated their expression in reactive astrocytes in human white matter injury (WMI). Functional studies on one of these genes revealed that mice lacking Asef exhibited impaired astrocyte differentiation during development and repair after WMI, coupled with compromised blood-brain barrier integrity in the adult CNS. These studies have identified distinct stages of astrocyte lineage development associated with human WMI and, together with our functional analysis of Asef, highlight the parallels between astrocyte development and their reactive counterparts associated with injury. SIGNIFICANCE STATEMENT: Astrocytes play a central role in CNS function and associated diseases. Yet the mechanisms that control their development remain poorly defined. Using the developing mouse spinal cord as a model system, we identify molecular changes that occur in developing astrocytes. These molecular signatures are strongly correlated with human astrocyte expression profiles and validation in mouse spinal cord identifies a host of new genes associated with the astrocyte lineage. These genes are present in reactive astrocytes in human white matter injury, and functional studies reveal that one of these genes, Asef, contributes to reactive astrocyte responses after injury. These studies identify distinct stages of astrocyte lineage development and highlight the parallels between astrocyte development and their reactive counterparts associated with injury.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Regeneração da Medula Espinal/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Troca de Nucleotídeo Guanina Rho , Fatores de TempoRESUMO
BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer in Japan. Smoking and drinking alcohol are environmental risk factors for ESCC, whereas single nucleotide polymorphisms in ADH1B and ALDH2, which increase harmful intermediates produced by drinking alcohol, are genetic risk factors. We conducted a large-scale genomic analysis of ESCCs from patients in Japan to determine the mutational landscape of this cancer. METHODS: We performed whole-exome sequence analysis of tumor and nontumor esophageal tissues collected from 144 patients with ESCC who underwent surgery at 5 hospitals in Japan. We also performed single-nucleotide polymorphism array-based copy number profile and germline genotype analyses of polymorphisms in ADH1B and ALDH2. Polymorphisms in CYP2A6, which increase harmful effects of smoking, were analyzed. Functions of TET2 mutants were evaluated in KYSE410 and HEK293FT cells. RESULTS: A high proportion of mutations in the 144 tumor samples were C to T substitution in CpG dinucleotides (called the CpG signature) and C to G/T substitutions with a flanking 5' thymine (called the APOBEC signature). Based on mutational signatures, patients were assigned to 3 groups, which associated with environmental (drinking and smoking) and genetic (polymorphisms in ALDH2 and CYP2A6) factors. Many tumors contained mutations in genes that regulate the cell cycle (TP53, CCND1, CDKN2A, FBXW7); epigenetic processes (MLL2, EP300, CREBBP, TET2); and the NOTCH (NOTCH1, NOTCH3), WNT (FAT1, YAP1, AJUBA) and receptor-tyrosine kinase-phosphoinositide 3-kinase signaling pathways (PIK3CA, EGFR, ERBB2). Mutations in EP300 and TET2 correlated with shorter survival times, and mutations in ZNF750 associated with an increased number of mutations of the APOBEC signature. Expression of mutant forms of TET2 did not increase cellular levels of 5-hydroxymethylcytosine in HEK293FT cells, whereas knockdown of TET2 increased the invasive activity of KYSE410 ESCC cells. Computational analyses associated the mutations in NFE2L2 we identified with transcriptional activation of its target genes. CONCLUSIONS: We associated environmental and genetic factors with base substitution patterns of somatic mutations and provide a registry of genes and pathways that are disrupted in ESCCs. These findings might be used to design specific treatments for patients with esophageal squamous cancers.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Genômica , Mutação , Polimorfismo de Nucleotídeo Único , Álcool Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial/genética , Povo Asiático/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/etnologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Ilhas de CpG , Citocromo P-450 CYP2A6/genética , Análise Mutacional de DNA , Neoplasias Esofágicas/etnologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Exoma , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Interação Gene-Ambiente , Estudos de Associação Genética , Predisposição Genética para Doença , Genômica/métodos , Células HEK293 , Humanos , Japão/epidemiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fatores de Risco , TransfecçãoRESUMO
Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.
Assuntos
Citoesqueleto de Actina/metabolismo , Permeabilidade da Membrana Celular , Endotélio Vascular/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Artéria Pulmonar/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Western Blotting , Células Cultivadas , Endotélio Vascular/citologia , Imunofluorescência , Humanos , Imunoprecipitação , Artéria Pulmonar/citologia , RNA Interferente Pequeno/genética , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Increased vascular endothelial permeability and inflammation are major pathological mechanisms of pulmonary edema and its life-threatening complication, the acute respiratory distress syndrome (ARDS). We have previously described potent protective effects of hepatocyte growth factor (HGF) against thrombin-induced hyperpermeability and identified the Rac pathway as a key mechanism of HGF-mediated endothelial barrier protection. However, anti-inflammatory effects of HGF are less understood. This study examined effects of HGF on the pulmonary endothelial cell (EC) inflammatory activation and barrier dysfunction caused by the gram-negative bacterial pathogen lipopolysaccharide (LPS). We tested involvement of the novel Rac-specific guanine nucleotide exchange factor Asef in the HGF anti-inflammatory effects. HGF protected the pulmonary EC monolayer against LPS-induced hyperpermeability, disruption of monolayer integrity, activation of NF-kB signaling, expression of adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and production of IL-8. These effects were critically dependent on Asef. Small-interfering RNA-induced downregulation of Asef attenuated HGF protective effects against LPS-induced EC barrier failure. Protective effects of HGF against LPS-induced lung inflammation and vascular leak were also diminished in Asef knockout mice. Taken together, these results demonstrate potent anti-inflammatory effects by HGF and delineate a key role of Asef in the mediation of the HGF barrier protective and anti-inflammatory effects. Modulation of Asef activity may have important implications in therapeutic strategies aimed at the treatment of sepsis and acute lung injury/ARDS-induced gram-negative bacterial pathogens.
Assuntos
Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Lesão Pulmonar/patologia , Lesão Pulmonar/fisiopatologia , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Inflamação/patologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein involved in transcription, mRNA splicing, mRNA stabilization and translation. Although hnRNP K has been suggested to play a role in the development of many cancers, its molecular function in colorectal cancer has remained elusive. Here we show that hnRNP K plays an important role in the mitotic process in HCT116 colon cancer cells. Furthermore, we demonstrate that hnRNP K directly transactivates the NUF2 gene, the product of which is a component of the NDC80 kinetochore complex and which is known to be critical for a stable spindle microtubule-kinetochore attachment. In addition, knockdown of both hnRNP K and NUF2 caused failure in metaphase chromosome alignment and drastic decrease in the growth of colon cancer cells. These results suggest that the hnRNP K-NUF2 axis is important for the mitotic process and proliferation of colon cancer cells and that this axis could be a target for the therapy of colon cancer.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Camundongos Endogâmicos BALB C , Mitose , Regiões Promotoras Genéticas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
LGR5 plays an important role in the self-renewal of stem cells and is used as a marker identifying self-renewing stem cells in small intestine and hair follicles. Moreover, LGR5 has been reported to be overexpressed in several cancers. SOX9 is a transcription factor that plays a key role in development, differentiation and lineage commitment in various tissues. It has also been reported that SOX9 is overexpressed in a variety of cancers and contributes to their malignant phenotype. Here we show that LGR5 is required for the tumorigenicity of glioblastoma cells. We further show that SOX9 is upregulated in glioblastoma cells and directly enhances the expression of LGR5. We also demonstrate that knockdown of SOX9 suppresses the proliferation and tumorigenicity of glioblastoma cells. These results suggest that SOX9-mediated transcriptional regulation of LGR5 is critical for the tumorigenicity of glioblastoma cells. We speculate that the SOX9-LGR5 pathway could be a potentially promising target for the therapy of glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Fatores de Transcrição SOX9/fisiologia , Regulação para Cima/fisiologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Humanos , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição SOX9/genéticaRESUMO
BACKGROUND: Lactobacillus gasseri SBT2055 (LG2055) has been shown to prevent abdominal adiposity, and suppression of lipid absorption is considered a possible mechanism, detail of which, however, are poorly understood. In the present study, we evaluated the effects of LG2055 on fat hydrolysis by determining pancreatic lipase activity and fat emulsion properties in vitro. We also examined whether LG2055 influences fecal fat excretion in humans. METHODS: Pancreatic lipase activity was investigated in vitro using an artificially prepared fat emulsion and 4-methylumbelliferyl oleate (4-MUO) as substrates. The concentrations of free fatty acids and 4-methylumbelliferone were quantified. Fat emulsion droplet size was measured using a particle size analyzer. The clinical study was performed as a double-blind, randomized, placebo-controlled trial. Subjects consumed 100 g of fermented milk (FM)/d, either with or without LG2055 supplementation, for seven days. Fecal samples were collected during three-day pre-observational and FM intake periods and fecal fat levels were determined. RESULTS: LG2055 dose-dependently suppressed lipase activity in the fat emulsion assay but not in the 4-MUO assay. LG2055 dose-dependently increased fat emulsion droplet size. The effects of LG2055 on lipase activity and fat emulsion properties were increased compared with four other tested strains (Lactobacillus gasseri SBT0317, Lactobacillus gasseri JCM1131T, Lactobacillus. delbrueckii subsp. bulgaricus JCM1002T and Streptococcus thermophilus ATCC19258T). In our clinical study, fecal fat level after FM intake was significantly increased compared with that observed before FM intake in the LG2055-containing active FM group but not the control FM group lacking LG2055. CONCLUSIONS: LG2055 increased fat emulsion droplet size, resulting in the suppression of lipase-mediated fat hydrolysis. The influence of LG2055 on the physicochemical properties of fat emulsion provides a mechanism for the probiotic-mediated suppression of lipid absorption and promotion of fecal fat excretion in humans. TRIAL REGISTRATION: UMIN000015772.
Assuntos
Gorduras/metabolismo , Ácidos Graxos/metabolismo , Fezes/química , Lactobacillus/metabolismo , Adulto , Idoso , Método Duplo-Cego , Emulsões/metabolismo , Gorduras/análise , Feminino , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Japão , Lipase/metabolismo , Masculino , Pessoa de Meia-Idade , Tamanho da PartículaRESUMO
Tumors are complex tissues composed of cancer cells, various types of stromal cells and extracellular matrix. Recent studies have shown that the tumor microenvironment(TME) plays an important role in both malignant tumor progression and treatment response. Thus, it is essential to improve our understanding of the mechanism of communication between cancer cells and their TME. Much effort has been devoted to developing agents that interfere with the interaction between cancer cells and their TME. In this review, we show a brief overview over the current knowledge about the TME-targeting agents and describe therapeutic strategies that have been designed to target components of the TME.
Assuntos
Terapia de Alvo Molecular , Neoplasias/etiologia , Neoplasias/terapia , Microambiente Tumoral , Animais , Progressão da Doença , Matriz Extracelular/fisiologia , Fibroblastos/fisiologia , Humanos , Hipóxia , Macrófagos/fisiologia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismoRESUMO
The tumor suppressor adenomatous polyposis coli (APC) is mutated in familial adenomatous polyposis and in many sporadic colorectal tumors. Adenomatous polyposis coli is known to negatively regulate Wnt signaling by inducing the degradation of ß-catenin. Adenomatous polyposis coli also interacts with the guanine nucleotide exchange factors Asef and Asef2 and stimulates their activity, thereby regulating cell adhesion and migration. Here we show that in confluent, non-motile MDCK II cells, Asef/Asef2 are colocalized with APC at the sites of cell-cell adhesion at the apical and junctional levels. In contrast, in colorectal tumor cells containing mutated APC, significant amounts of Asef/Asef2 and the truncated mutant APCs are localized mainly in the cytoplasm. These results suggest that localization of the Asef/Asef2-APC complex at the sites of cell-cell contact is critical for the regulation of cell adhesion, and that the aberrant subcellular localization of these complexes in colorectal tumor cells may contribute to the cell's aberrant adhesive and migratory properties.
Assuntos
Polipose Adenomatosa do Colo/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Cultivadas , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citoplasma/metabolismo , Citoplasma/fisiologia , Cães , Células Madin Darby de Rim Canino , MutaçãoRESUMO
Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of beta-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef(-/-) mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Movimento Celular , Neoplasias Colorretais/mortalidade , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neovascularização Patológica/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Neoplasias Colorretais/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The tumour suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC binds to beta-catenin, a key component of the Wnt signalling pathway, and induces its degradation. APC interacts with microtubules and accumulates at their plus ends in membrane protrusions, and associates with the plasma membrane in an actin-dependent manner. In addition, APC interacts with the Rac-specific guanine nucleotide exchange factor Asef and stimulates its activity, thereby regulating the actin cytoskeletal network and cell morphology. Here we show that overexpression of Asef decreases E-cadherin-mediated cell-cell adhesion and promotes the migration of epithelial Madin-Darby canine kidney cells. Both of these activities are stimulated by truncated APC proteins expressed in colorectal tumour cells. Experiments based on RNA interference and dominant-negative mutants show that both Asef and mutated APC are required for the migration of colorectal tumour cells expressing truncated APC. These results suggest that the APC-Asef complex functions in cell migration as well as in E-cadherin-mediated cell-cell adhesion, and that truncated APC present in colorectal tumour cells contributes to their aberrant migratory properties.
Assuntos
Movimento Celular/genética , Neoplasias Colorretais/patologia , Genes APC , Fatores de Troca do Nucleotídeo Guanina/genética , Mutação , Animais , Sequência de Bases , Western Blotting , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Primers do DNA , Cães , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Testes de Precipitina , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of beta-catenin, through its interaction with beta-catenin, GSK-3 beta and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A-KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and beta-catenin are transported along microtubules by KAP3-KIF3A-KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Cinesinas/metabolismo , Proteínas Repressoras , Animais , Proteína Axina , Encéfalo/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Cães , Células Epiteliais/metabolismo , Biblioteca Gênica , Quinase 3 da Glicogênio Sintase , Histonas/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas de Protozoários/metabolismo , Transativadores/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus , Xenopus laevis , beta CateninaRESUMO
Sporadic and familial colorectal tumours usually harbour biallelic adenomatous polyposis coli (APC)-associated mutations that result in constitutive activation of Wnt signalling. Furthermore, APC activates Asef and Asef2, which are guanine-nucleotide exchange factors specific for Rac1 and Cdc42. Here, we show that Asef and Asef2 expression is aberrantly enhanced in intestinal adenomas and tumours. We also show that deficiency of either Asef or Asef2 significantly reduces the number and size of adenomas in Apc(Min/+) mice, which are heterozygous for an APC mutation and spontaneously develop adenomas in the intestine. We observed that the APC-Asef/Asef2 complex induces c-Jun amino-terminal kinase-mediated transactivation of matrix metalloproteinase 9, and is required for the invasive activity of colorectal tumour cells. Furthermore, we show that Asef and Asef2 are required for tumour angiogenesis. These results suggest that Asef and Asef2 have a crucial role in intestinal adenoma formation and tumour progression, and might be promising molecular targets for the treatment of colorectal tumours.
Assuntos
Adenoma/genética , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Genes APC , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Neoplasias Intestinais/genética , Adenoma/patologia , Animais , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Genes APC/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Células HCT116 , Humanos , Neoplasias Intestinais/patologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/genética , Células Tumorais CultivadasRESUMO
Numerous independent clinical and experimental studies indicate that estrogens confer a protective effect against development of intestinal tumors, however the molecular mechanisms involved remain unclear. Physiological effects of estrogens are predominantly mediated by the action of nuclear estrogen receptors (ERs). A multifunctional protein adenomatous polyposis coli (APC) is a tumor suppressor and thought to act as a gatekeeper in colon tumorigenesis, as loss of function APC mutations trigger the development of colorectal cancer. Here we report that APC physically associates with ERa in the ligand-dependent manner. We have shown in the endogenous setting that the ligand-activated ERa recruits APC to the promoters in ER target genes and that increased levels of ER-dependent recruitment of APC enhances the ER transactivation through stimulation of histone acetylation. Found in majority of human colon tumors APC truncation mutants lost the ability to interact with ER. Thus, here we present the first evidence of a functional interaction between APC and ER that may be accounted for a tumor protective action of estrogens.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Sequência de Aminoácidos , Estrogênios/fisiologia , Genes APC , Células HCT116 , Humanos , Ligantes , Elementos de Resposta/fisiologia , Deleção de Sequência , Ativação Transcricional/fisiologiaRESUMO
The Wnt signaling pathway is aberrantly activated in the majority of human colorectal tumors. ß-catenin, a key component of the Wnt signaling pathway, interacts with the T-cell factor/lymphoid enhancer-binding factor family of transcription factors and activates transcription of Wnt target genes. Sp5 is one of the Wnt target genes, and its expression is commonly upregulated in colon cancer cells. The present study demonstrates that the expression of Sp5 is not upregulated in the colon cancer cell line HCT116, in which Wnt signaling is constitutively activated. Furthermore, the results demonstrate that Sp5 has the potential to inhibit cell proliferation through upregulation of the cell cycle inhibitor p27. These findings suggest that HCT116 cells downregulate Sp5 to avoid p27-mediated growth arrest.
RESUMO
Aberrant activation of Wnt/ß-catenin signaling is a major driving force in colon cancer. Wnt/ß-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc regulates multiple biological processes through its ability to directly modulate gene expression. Here, we identify a direct target of c-Myc, termed MYU, and show that MYU is upregulated in most colon cancers and required for the tumorigenicity of colon cancer cells. Furthermore, we demonstrate that MYU associates with the RNA binding protein hnRNP-K to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. These results suggest that the MYU/hnRNP-K/CDK6 pathway functions downstream of Wnt/c-Myc signaling and plays a critical role in the proliferation and tumorigenicity of colon cancer cells.
Assuntos
Ciclo Celular , Quinase 6 Dependente de Ciclina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/metabolismo , Via de Sinalização Wnt , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Camundongos Nus , RNA Longo não Codificante/genética , Regulação para Cima/genéticaRESUMO
Increased levels of hepatocyte growth factor (HGF) in injured lungs may reflect a compensatory response to diminish acute lung injury (ALI). HGF-induced activation of Rac1 GTPase stimulates endothelial barrier protective mechanisms. This study tested the involvement of Rac-specific guanine nucleotide exchange factor Asef in HGF-induced endothelial cell (EC) cytoskeletal dynamics and barrier protection in vitro and in a two-hit model of ALI. HGF induced membrane translocation of Asef and stimulated Asef Rac1-specific nucleotide exchange activity. Expression of constitutively activated Asef mutant mimicked HGF-induced peripheral actin cytoskeleton enhancement. In contrast, siRNA-induced Asef knockdown or expression of dominant-negative Asef attenuated HGF-induced Rac1 activation evaluated by Rac-GTP pull down and FRET assay with Rac1 biosensor. Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability. Intravenous HGF injection attenuated lung inflammation and vascular leak in the two-hit model of ALI induced by excessive mechanical ventilation and thrombin signaling peptide TRAP6. This effect was lost in Asef(-/-) mice. This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.
Assuntos
Permeabilidade da Membrana Celular , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Pulmão/metabolismo , Animais , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Camundongos , Interferência de RNA , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The transcription factor GATA6 is a critical regulator of cell proliferation and development in the gastrointestinal tract. We have recently reported that GATA6 induces the expression of the intestinal stem cell marker LGR5 and enhances the clonogenicity and tumorigenicity of colon cancer cells, but not the growth of these cells cultured under adherent conditions. Here we show that REG4, a member of the regenerating islet-derived (REG) family, is also a target of GATA6. We further demonstrate that REG4 is downregulated by overexpression of miR-363, which suppresses GATA6 expression. Moreover, we show that GATA6-mediated activation of REG4 enhances the growth of colon cancer cells under adherent conditions and is required for their tumorigenicity. Taken together, our findings demonstrate that GATA6 simultaneously induces the expression of genes essential for the growth of colon cancer cells under adherent conditions (REG4) and genes required for their clonogenicity (LGR5), and that the miR-363-GATA6-REG4/LGR5 signaling cascade promotes the tumorigenicity of colon cancer cells.