RESUMEN
The stability of a protein, as well as its function and versatility, can be enhanced through oligomerization. KITENIN (KAI1 C-terminal interacting tetraspanin) is known to promote the malignant progression of colorectal cancer (CRC). How KITENIN maintains its structural integrity and stability are largely unknown, however. Here we investigated the mechanisms regulating the stability of KITENIN with the aim of developing therapeutics blocking its oncogenic functions. We found that KITENIN formed a homo-oligomeric complex and that the intracellular C-terminal domain (KITENIN-CTD) was needed for this oligomerization. Expression of the KITENIN-CTD alone interfered with the formation of the KITENIN homodimer, and the amino acid sequence from 463 to 471 within the KITENIN-CTD was the most effective. This sequence coupled with a cell-penetrating peptide was named a KITENIN dimerization-interfering peptide (KDIP). We next studied the mechanisms by which KDIP affected the stability of KITENIN. The KITENIN-interacting protein myosin-X (Myo10), which has oncogenic activity in several cancers, functioned as an effector to stabilize the KITENIN homodimer in the cis formation. Treatment with KDIP resulted in the disintegration of the homodimer via downregulation of Myo10, which led to increased binding of RACK1 to the exposed RACK1-interacting motif (463-471 aa), and subsequent autophagy-dependent degradation of KITENIN and reduced CRC cell invasion. Intravenous injection of KDIP significantly reduced the tumour burden in a syngeneic mouse tumour model and colorectal liver metastasis in an intrasplenic hepatic metastasis model. Collectively, our present results provide a new cancer therapeutic peptide for blocking colorectal liver metastasis, which acts by inducing the downregulation of Myo10 and specifically targeting the stability of the oncogenic KITENIN protein.
Asunto(s)
Neoplasias Colorrectales , Proteínas de la Membrana , Péptidos , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Dimerización , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/secundario , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Miosinas/química , Miosinas/metabolismo , Proteínas Oncogénicas/química , Proteínas Oncogénicas/metabolismo , Péptidos/farmacología , Estabilidad Proteica/efectos de los fármacosRESUMEN
Fibroblast growth factor receptor 4 (FGFR4) is known to induce cancer cell proliferation, invasion, and antiapoptosis through activation of RAS/RAF/ERK and PI3K/AKT pathways, which are also known as major molecular bases of colon cancer carcinogenesis related with epidermal growth factor receptor (EGFR) signaling. However, the interaction between FGFR4 and EGFR signaling in regard to colon cancer progression is unclear. Here, we investigated a potential cross-talk between FGFR4 and EGFR, and the effect of anti-EGFR therapy in colon cancer treatment. To explore the biological roles of FGFR4 in cancer progression, RNA sequencing was carried out using FGFR4 transfected colon cell lines. Gene ontology data showed the upregulation of genes related to EGFR signaling, and we identified that FGFR4 overexpression secretes EGFR ligands such as amphiregulin (AREG) with consequent activation of EGFR and ErbB3. This result was also shown in in vivo study and the cooperative interaction between EGFR and FGFR4 promoted tumor growth. In addition, FGFR4 overexpression reduced cetuximab-induced cytotoxicity and the combination of FGFR4 inhibitor (BLU9931) and cetuximab showed profound antitumor effect compared to cetuximab alone. Clinically, we found the positive correlation between FGFR4 and AREG expression in tumor tissue, but not in normal tissue, from colon cancer patients and these expressions were significantly correlated with poor overall survival in patients treated with cetuximab. Therefore, our results provide the novel mechanism of FGFR4 in connection with EGFR activation and the combination of FGFR4 inhibitor and cetuximab could be a promising therapeutic option to achieve the optimal response to anti-EGFR therapy in colon cancer.
Asunto(s)
Anfirregulina/genética , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Línea Celular Tumoral , Cetuximab/farmacología , Neoplasias del Colon/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Galectin-3-binding protein (Gal-3BP) is a member of the family of scavenger receptor cysteine-rich (SRCR) domain-containing proteins, which are associated with the immune system. However, the functional roles and signaling mechanisms of Gal-3BP in host defense and the immune response remain largely unknown. Here, we identified cellular Gal-3BP as a negative regulator of NF-κB activation and proinflammatory cytokine production in lipopolysaccharide (LPS)-stimulated murine embryonic fibroblasts (MEFs). Furthermore, cellular Gal-3BP interacted with transforming growth factor ß-activated kinase 1 (TAK1), a crucial mediator of NF-κB activation in response to cellular stress. Gal-3BP inhibited the phosphorylation of TAK1, leading to suppression of its kinase activity and reduced protein stability. In vivo we found that Lgals3BP deficiency in mice enhanced LPS-induced proinflammatory cytokine release and rendered mice more sensitive to LPS-induced endotoxin shock. Overall, these results suggest that Gal-3BP is a novel suppressor of TAK1-dependent NF-κB activation that may have potential in the prevention and treatment of inflammatory diseases.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Animales , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular , Citocinas/metabolismo , Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Unión Proteica , ProteolisisRESUMEN
Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/ß-catenin signaling by Wnt8 is especially critical. Glycoproteins of the Dickkopf (Dkk) family are important modulators of Wnt signaling acting primarily as Wnt antagonists. Here we report that Dkk2 is required for neural crest specification functioning as a positive regulator of Wnt/ß-catenin signaling. Dkk2 depletion in Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or ß-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and ß-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and ß-catenin, however unlike Wnt8, in a GSK3ß independent manner. These findings suggest that Wnt8 and Dkk2 converge on ß-catenin using distinct transduction pathways both independently required to activate Wnt/ß-catenin signaling and induce neural crest cells.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Cresta Neural/fisiología , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismo , Animales , Linaje de la Célula , Células Cultivadas , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Glucógeno Sintasa Quinasa 3 beta/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Cresta Neural/citología , Vía de Señalización Wnt , Proteínas de Xenopus/genética , Xenopus laevis , beta Catenina/genéticaRESUMEN
Hes3 belongs to the Hes basic helix-loop-helix family of transcriptional repressors that play central roles in maintaining progenitor cells and regulating binary cell fate decisions in the embryo. During Xenopus laevis development, hes3 is expressed in the embryonic ectoderm in a horseshoe shape domain at the edge of the developing neural pate. Hes3 mis-expression at early neurula stage blocks neural crest (snai2, sox8, sox9 and sox10) and cranial placode (six1 and dmrta1) gene expression, and promotes neural plate (sox2 and sox3) fate. At tailbud stage, these embryos exhibited a massive up-regulation of both sox8 and sox10 expression, associated with an increase in genes important for melanocytes differentiation (mitf and dct). Using a hormone inducible construct we show that Hes3 does not induce a pigment cell differentiation program de novo, rather it maintains progenitor cells in an undifferentiated state, and as Hes3 expression subsides overtime these cells adopt a pigment cell fate. We demonstrate that mechanistically Hes3 mediates its activity through inhibition of Wnt/ß-catenin signaling, a molecular pathway critical for neural crest specification and pigment cell lineage differentiation. We propose that Hes3 at the edge of the neural plate spatially restricts the response to mesoderm-derived Wnt ligands, thereby contributing to the establishment of sharp boundaries of gene expression at the neural plate border.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Desarrollo Embrionario , Secuencias Hélice-Asa-Hélice , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Placa Neural/citología , Filogenia , Factores de Transcripción SOXB1/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
p73 is a member of the p53 family of transcription factors and, like p53, plays a role as a tumor suppressor. p73 is involved in development, proliferation, apoptosis and metastasis. However, the precise molecular mechanisms underlying its function in inhibiting metastasis remain largely unknown. Here, we show that induction of TAp73 decreased invasion and migration activity of colorectal cancer cells, whereas knockdown of TAp73 led to increased invasion and migration activity. KAI1 was identified as a transcriptional target of TAp73 and its expression is indispensable for TAp73-mediated inhibition of cell invasion and migration. Furthermore, induction of TAp73 in colorectal cancer cells elevated KAI1 expression and decreased the frequency of hepatic metastasis in vivo. Whereas, the decreased invasion and migration activities caused by TAp73 induction were abrogated by knockdown of KAI1. Interestingly, TAp73 and KAI1 are overexpressed in primary colorectal cancers and a significant correlation between TAp73 and KAI1 expression was detected, but their expressions were significantly down-regulated in metastatic cancers. Taken together, our results support a novel role for TAp73 in controlling colorectal cancer cell invasion, migration and metastasis by regulating transcription of KAI1.
Asunto(s)
Movimiento Celular/genética , Neoplasias Colorrectales/genética , Proteínas de la Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Proteína Tumoral p73/genética , Animales , Células CACO-2 , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Doxiciclina/farmacología , Proteínas de la Matriz Extracelular/metabolismo , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Invasividad Neoplásica , Proteínas del Tejido Nervioso/metabolismo , Proteína Tumoral p73/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
During embryogenesis vertebrates develop a complex craniofacial skeleton associated with sensory organs. These structures are primarily derived from two embryonic cell populations the neural crest and cranial placodes, respectively. Neural crest cells and cranial placodes are specified through the integrated action of several families of signaling molecules, and the subsequent activation of a complex network of transcription factors. Here we describe the expression and function of Anosmin-1 (Anos1), an extracellular matrix protein, during neural crest and cranial placodes development in Xenopus laevis. Anos1 was identified as a target of Pax3 and Zic1, two transcription factors necessary and sufficient to generate neural crest and cranial placodes. Anos1 is expressed in cranial neural crest progenitors at early neurula stage and in cranial placode derivatives later in development. We show that Anos1 function is required for neural crest and sensory organs development in Xenopus, consistent with the defects observed in Kallmann syndrome patients carrying a mutation in ANOS1. These findings indicate that anos1 has a conserved function in the development of craniofacial structures, and indicate that anos1-depleted Xenopus embryos represent a useful model to analyze the pathogenesis of Kallmann syndrome.
Asunto(s)
Desarrollo Embrionario/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismo , Neurogénesis/fisiología , Cráneo/embriología , Cráneo/metabolismo , Proteínas de Xenopus/metabolismo , Animales , XenopusRESUMEN
The transcription factors Pax3 and Zic1 are critical to specify the neural plate border and to promote neural crest formation. In a microarray screen designed to identify genes regulated by Pax3 and Zic1 in Xenopus we isolated Znf703/Nlz1 a transcriptional repressor member of the NET (NocA/Nlz, Elbow, and TLP-1) protein family. At early neurula stage znf703 is expressed in the dorsal ectoderm, spanning the neural plate and neural plate border, with an anterior boundary of expression corresponding to rhombomeres 3 and 4 (r3/r4) in the prospective hindbrain. As a bonafide target of Pax3 and Zic1, znf703 is activated by neural plate border inducing signals, and its expression depends on Pax3 and Zic1 function in the embryo. Znf703 morpholino-mediated knockdown expanded several posterior hindbrain genes, while Znf703 overexpression completely obliterated the expression of these segmental genes, signifying that the transcriptional repressor activity of Znf703 is critical to pattern the hindbrain. Furthermore, snai2 and sox10 expression was severely impaired upon manipulation of Znf703 expression levels in the embryo suggesting that Znf703 participates in neural crest formation downstream of Pax3 and Zic1 in Xenopus.
Asunto(s)
Cresta Neural/crecimiento & desarrollo , Neurogénesis/genética , Proteínas Nucleares/genética , Factor de Transcripción PAX3/genética , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Morfolinos/genética , Cresta Neural/metabolismo , Rombencéfalo/crecimiento & desarrollo , Rombencéfalo/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción de la Familia Snail/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
PURPOSE: Fibroblast growth factor receptor 4 (FGFR4) plays an important role in cancer progression during tumor proliferation, invasion, and metastasis. This study evaluated the prognostic role of FGFR4 polymorphism in patients with resected colon cancer, including the underlying mechanism. MATERIALS AND METHODS: FGFR4 polymorphism was characterized in patientswho received curative resection for stage III colon cancer. FGFR4-dependent signal pathways involving cell proliferation, invasion, and migration according to genotypes were also evaluated in transfected colon cancer cell lines. RESULTS: Among a total of 273 patients, the GG of FGFR4 showed significantly better overall survival than the AG or AA, regardless of adjuvant treatment. In the group of AG or AA, combination of folinic acid, fluorouracil, and oxaliplatin (FOLFOX) resulted in better survival than fluorouracil/leucovorin or no adjuvant chemotherapy. However, in GG, there was no difference among treatment regimens. Using multivariate analyses, the Arg388 carriers, together with age, N stage, poor differentiation, absence of a lymphocyte response, and no adjuvant chemotherapy, had a significantly worse OS than patients with the Gly388 allele. In transfected colon cancer cells, overexpression of Arg388 significantly increased cell proliferation and changes in epithelial to mesenchymal transition markers compared with cells overexpressing the Gly388 allele. CONCLUSION: The Arg388 allele of FGFR4 may be a biomarker and a candidate target for adjuvant treatment of patients with resected colon cancer.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias del Colon/mortalidad , Transición Epitelial-Mesenquimal/genética , Mutación , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Sustitución de Aminoácidos , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Quimioterapia Adyuvante , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Expresión Génica , Genotipo , Humanos , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Estadificación de Neoplasias , Polimorfismo Genético , Pronóstico , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause of Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4.
Asunto(s)
Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica/genética , Disostosis Mandibulofacial/genética , Desarrollo Maxilofacial/genética , Factores de Empalme de ARN/genética , Proteínas de Xenopus/deficiencia , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Región Branquial/embriología , Región Branquial/metabolismo , Región Branquial/patología , Cartílago/crecimiento & desarrollo , Cartílago/metabolismo , Cartílago/patología , Codón sin Sentido , Mutación del Sistema de Lectura , Técnicas de Silenciamiento del Gen , Prueba de Complementación Genética , Humanos , Disostosis Mandibulofacial/embriología , Disostosis Mandibulofacial/fisiopatología , Datos de Secuencia Molecular , Morfolinos/farmacología , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Placa Neural/embriología , Placa Neural/patología , Fenotipo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Cráneo/anomalías , Cráneo/embriología , Cráneo/crecimiento & desarrollo , Proteínas de Xenopus/biosíntesis , Proteínas de Xenopus/genética , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Xenopus laevis/crecimiento & desarrolloRESUMEN
HOX (homeobox) genes encode a family of transcriptional regulators, which have an important role in morphogenesis and differentiation during embryonic development. Their deregulated expression is involved in the carcinogenesis of many human solid tumours. In the present study, we show that HOXB5 mRNA was significantly overexpressed in gastric cancer tissues compared with adjacent normal tissues. HOXB5-up-regulated cancer cells showed increased invasion and migration activity, but no change in proliferation activity, whereas HOXB5-down-regulated cells showed decreased invasion and migration activity. Up-regulation of HOXB5 resulted in up-regulation of ß-catenin, whereas inhibition of HOXB5 expression by siRNA led to the down-regulation of ß-catenin. Moreover, a significant correlation between HOXB5 and CTNNB1 (ß-catenin) mRNA expression was detected in gastric cancer tissues. Furthermore, we found that HOXB5 binds directly to the CTNNB1 promoter region and activates the transcriptional expression of ß-catenin, as well as its downstream target genes, encoding cyclin D1 and c-Myc, leading to an increase in the invasion and migration activity of human gastric cancer cells. Thus HOXB5 may be an important regulator of the Wnt/ß-catenin signalling pathway, thereby contributing to gastric cancer progression and metastasis.
Asunto(s)
Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Neoplasias Gástricas/metabolismo , Transcripción Genética , Regulación hacia Arriba , Línea Celular Tumoral , Proliferación Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas de Homeodominio/genética , Humanos , Invasividad Neoplásica , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Vía de Señalización Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas aeruginosa, termed Paz, demonstrates anticancer activity against a range of cancers but not against brain tumors. In contrast, Laz is produced by members of Gonococci/Meningococci, including Neisseria meningitides which can cross the blood-brain barrier to infect brain meninges. It has been previously reported that Laz has an additional 39 amino acid moiety, called an H.8 epitope, in the N-terminal part of the azurin moiety that allows Laz to cross the entry barrier to brain tumors such as glioblastomas. Exactly, how the H.8 epitope helps the azurin moiety of Laz to cross the entry barriers to attack glioblastoma cells is unknown. In this paper, we describe the structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a ß-sandwich fold with 2 ß-sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity.
Asunto(s)
Antineoplásicos/farmacología , Azurina/farmacología , Proteínas Bacterianas/farmacología , Neisseria/genética , Secuencia de Aminoácidos , Antineoplásicos/química , Azurina/química , Proteínas Bacterianas/química , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Datos de Secuencia Molecular , Neisseria/metabolismo , Conformación Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Difracción de Rayos XRESUMEN
Transcription factors Pax3 and Zic1 are among the earliest genes activated at the neural plate border. In Xenopus, they are necessary and sufficient to promote the formation of multiple neural plate border cell types, including the neural crest, cranial placodes, and hatching gland. Pax3 is especially critical for the formation of the hatching gland, a group of cells that produce proteolytic enzymes essential to digest the egg vitelline envelope and jelly coat in order to release the tadpole into the environment. In a screen designed to identify downstream targets of Pax3, we isolated a member of the astacin family of metalloproteases, related to Xenopus hatching enzyme (Xhe), that we named Xhe2. Xhe2 is exclusively expressed in hatching gland cells as they first emerge at the lateral edge of the anterior neural plate, and persists in this tissue up to the tadpole stage. Knockdown experiments show that Xhe2 expression depends entirely on Pax3 function. Gain-of-function studies demonstrate that Pax3 can induce premature hatching through the upregulation of several proteolytic enzymes including Xhe2. Interestingly, Xhe2 overexpression is sufficient to induce early hatching, indicating that Xhe2 is one of the key components of the degradation mechanism responsible for breaking down the vitelline membrane.
Asunto(s)
Metaloendopeptidasas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/crecimiento & desarrollo , Animales , Clonación Molecular , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Metaloendopeptidasas/genética , Placa Neural/metabolismo , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Membrana Vitelina/metabolismo , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
Thymosin ß4 (Tß4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tß4 in cell invasion and migration remain unclear. In this study, we show that Tß4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tß4 were correlated with stage of colorectal cancer, and that Tß4 expression was associated with morphogenesis and EMT. Tß4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tß4-downregulated cells showed decreased activities. We also demonstrated that Tß4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3ß, the expression and nuclear localization of ß-catenin, and integrin receptor activation. These results suggest that Tß4 is an important regulator of the ILK/AKT/ß-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.
Asunto(s)
Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Timosina/genética , beta Catenina/genética , Anciano , Línea Celular Tumoral , Movimiento Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal , Femenino , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Integrinas/genética , Integrinas/metabolismo , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Timosina/metabolismo , Microambiente Tumoral , beta Catenina/metabolismoRESUMEN
Transcription factors Pax3 and Zic1 are two important regulators of cell fate decision at the neural plate border, where they act synergistically to promote neural crest (NC) formation. To understand the role of these factors in NC development, we performed a microarray analysis to identify downstream targets of Pax3 and Zic1 in Xenopus embryos. Among the genes identified was a member of transcription factor activator protein 2 (Tfap2) family, Tfap2 epsilon (Tfap2e). Tfap2e is first expressed at early neurula stage in NC progenitors and Rohon-Beard sensory neurons, and persists in a subset of migrating cranial NC cells as they populate the pharyngeal arches. This is in contrast to other species in which Tfap2e is not detected in the early NC lineage. Tfap2e morpholino-mediated knockdown results in a loss of NC progenitors and an expansion of the neural plate. Tfap2e is also sufficient to activate NC-specific genes in animal cap explants, and gain-of-function experiments in the whole embryo indicate that Tfap2e can promote NC formation. We propose that Tfap2e is a novel player in the gene regulatory network controlling NC specification in Xenopus downstream of Pax3 and Zic1.
Asunto(s)
Cresta Neural/crecimiento & desarrollo , Cresta Neural/fisiología , Placa Neural/fisiología , Factor de Transcripción AP-2/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hibridación in Situ , Análisis por Micromatrices , Morfolinos , Células-Madre Neurales/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Receptoras Sensoriales/fisiología , Homología de Secuencia , Xenopus laevisRESUMEN
The neural crest (NC) is a multipotent population of migratory cells unique to the vertebrate embryo, contributing to the development of multiple organ systems. Transcription factors pax3 and zic1 are among the earliest genes activated in NC progenitors, and they are both necessary and sufficient to promote NC fate. In order to further characterize the function of these transcription factors during NC development we have used hormone inducible fusion proteins in a Xenopus animal cap assay, and DNA microarray to identify downstream targets of Pax3 and Zic1. Here we present the results of this screen and the initial validation of these targets using quantitative RT-PCR, in situ hybridization and morpholinos-mediated knockdown. Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. We also obtained several factors with no known function in Xenopus NC, which represent novel regulators of NC fate. The comprehensive characterization of Pax3 and Zic1 targets function in the NC gene regulatory network, are essential to understanding the mechanisms regulating the emergence of this important cell population.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Cresta Neural/embriología , Factores de Transcripción Paired Box/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes/fisiología , Hibridación in Situ , Análisis por Micromatrices , Morfolinos/genética , Cresta Neural/metabolismo , Factor de Transcripción PAX3 , Reacción en Cadena en Tiempo Real de la Polimerasa , Xenopus laevis/genéticaRESUMEN
BACKGROUND: Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3, and foxn1, and evaluated the neural crest contribution to thymus development. RESULTS: In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T-cell differentiation in frogs. CONCLUSIONS: These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Cresta Neural/embriología , Organogénesis/fisiología , Timo/embriología , Factores de Transcripción/metabolismo , Xenopus laevis/embriología , Animales , Secuencia de Bases , Cartilla de ADN/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia , Especificidad de la Especie , Timo/anatomía & histología , Factores de Transcripción/genéticaRESUMEN
Lower vertebrates develop a unique set of primary sensory neurons located in the dorsal spinal cord. These cells, known as Rohon-Beard (RB) sensory neurons, innervate the skin and mediate the response to touch during larval stages. Here we report the expression and function of the transcription factor Xaml1/Runx1 during RB sensory neurons formation. In Xenopus embryos Runx1 is specifically expressed in RB progenitors at the end of gastrulation. Runx1 expression is positively regulated by Fgf and canonical Wnt signaling and negatively regulated by Notch signaling, the same set of factors that control the development of other neural plate border cell types, i.e. the neural crest and cranial placodes. Embryos lacking Runx1 function fail to differentiate RB sensory neurons and lose the mechanosensory response to touch. At early stages Runx1 knockdown results in a RB progenitor-specific loss of expression of Pak3, a p21-activated kinase that promotes cell cycle withdrawal, and of N-tub, a neuronal-specific tubulin. Interestingly, the pro-neural gene Ngnr1, an upstream regulator of Pak3 and N-tub, is either unaffected or expanded in these embryos, suggesting the existence of two distinct regulatory pathways controlling sensory neuron formation in Xenopus. Consistent with this possibility Ngnr1 is not sufficient to activate Runx1 expression in the ectoderm. We propose that Runx1 function is critically required for the generation of RB sensory neurons, an activity reminiscent of that of Runx1 in the development of the mammalian dorsal root ganglion nociceptive sensory neurons.
Asunto(s)
Gástrula/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Receptoras Sensoriales/fisiología , Médula Espinal/citología , Factores de Transcripción/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Técnicas de Silenciamiento del Gen , Hibridación in Situ , Morfolinos/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Células Receptoras Sensoriales/citología , Médula Espinal/embriología , Proteínas de Xenopus/metabolismoRESUMEN
TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid and other cancer tissues, although its oncogenic significance and molecular mechanisms are unknown. Previously, we have shown that TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial-mesenchymal transition (EMT). In this study, we explored the molecular basis underlying TMPRSS4-mediated effects. We show that multiple downstream signaling pathways, including focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), Akt, Src and Rac1, are activated by TMPRSS4 expression and that FAK signaling and ERK activation are required for TMPRSS4-induced invasiveness and EMT, including cadherin switch. Inhibition of PI3K or Src reduced invasiveness and actin rearrangement mediated by TMPRSS4 without restoring E-cadherin expression. Downregulation of E-cadherin was required for TMPRSS4-mediated effects but was not sufficient to induce EMT and invasion. TMPRSS4 induced integrin alpha5 expression and its signal transduction, leading to invasiveness and EMT accompanied by downregulation of E-cadherin. Functional blocking confirmed that integrin alpha5beta1 is a critical signaling molecule that is sufficient to induce TMPRSS4-mediated effects. Immunohistochemical analysis showed that TMPRSS4 expression was significantly higher in human colorectal cancer tissues from advanced stages than in that of early stage. Furthermore, upregulation of TMPRSS4 was correlated with enhanced integrin alpha5 expression. These observations implicate integrin alpha5 upregulation as a molecular mechanism by which TMPRSS4 induces invasion and contributes to cancer progression.
Asunto(s)
Neoplasias Colorrectales/patología , Células Epiteliales/patología , Integrina alfa5/fisiología , Proteínas de la Membrana/fisiología , Mesodermo/patología , Serina Endopeptidasas/fisiología , Transducción de Señal/fisiología , Cadherinas/fisiología , Diferenciación Celular , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Humanos , Invasividad Neoplásica , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Chemokines are a family of proteins originally identified for their activity promoting the recruitment of leukocytes to inflammatory sites. Recent evidence indicates that chemokines and their receptors may also regulate key developmental processes. In this paper we report the expression and regulation of the chemokine CXCL14 during Xenopus laevis embryogenesis. CXCL14 is first detected in several ectoderm derivatives, the dorsal aspect of the retina, the cement gland and the hatching gland. Later in development, additional domains of expression include the head mesenchyme and the medial ventral aspect of the otic vesicle. CXCL14 expression in the ectoderm is regulated by both Bmp and canonical Wnt signaling. In the hatching gland CXCL14 is co-expressed with the transcription factor Pax3. Using gain of function and knockdown approaches in whole embryos and animal explants we show that Pax3 is both necessary and sufficient for CXCL14 expression in this domain of the ectoderm.