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1.
Exp Cell Res ; 353(2): 88-99, 2017 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-28284838

RESUMEN

In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin ß1, which is the most highly expressed integrin in our cohort of uveal melanomas. We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin ß1 on collagen I. Knockdown experiments confirmed integrin ß1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin ß1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin ß1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin ß1 in FAs on collagen I but not on fibronectin. This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.


Asunto(s)
Movimiento Celular/genética , Integrina beta1/biosíntesis , Melanoma/genética , Proteínas de Neoplasias/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Neoplasias de la Úvea/genética , Adhesión Celular/genética , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/patología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Integrina beta1/genética , Melanoma/patología , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatasas/genética , Neoplasias de la Úvea/patología
3.
Nature ; 480(7375): 94-8, 2011 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-22012259

RESUMEN

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.


Asunto(s)
Carcinoma de Células Renales/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Movimiento Celular/genética , Frecuencia de los Genes , Humanos , Invasividad Neoplásica/genética , Sumoilación
4.
J Neurosci ; 34(4): 1530-41, 2014 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-24453340

RESUMEN

Retinal progenitor proliferation and differentiation are tightly controlled by extrinsic cues and distinctive combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we have characterized Bcl-2-associated transcription factor (Bclaf1) during rodent retinogenesis. Bclaf1 expression is restricted to early-born cell types, such as ganglion, amacrine, and horizontal cells. Analysis of developing retinas in Bclaf1-deficient mice revealed a reduction in the numbers of retinal ganglion cells, amacrine cells and horizontal cells and an increase in the numbers of cone photoreceptor precursors. Silencing of Bclaf1expression by in vitro electroporation of shRNA in embryonic retina confirmed that Bclaf1 serves to promote amacrine and horizontal cell differentiation. Misexpression of Bclaf1 in late retinal progenitors was not sufficient to directly induce the generation of amacrine and horizontal cells. Domain deletion analysis indicated that the N-terminal domain of Bclaf1 containing an arginine-serine-rich and a bZip domain is required for its effects on retinal cell differentiation. In addition, analysis revealed that Bclaf1 function occurs independently of its interaction with endogenous Bcl-2-related proteins. Altogether, our data demonstrates that Bclaf1expression in postmitotic early-born cells facilitates the differentiation of early retinal precursors into retinal ganglion cells, amacrine cells, and horizontal cells rather than into cone photoreceptors.


Asunto(s)
Diferenciación Celular/fisiología , Células-Madre Neurales/citología , Neurogénesis/fisiología , Proteínas Represoras/metabolismo , Neuronas Retinianas/citología , Neuronas Retinianas/metabolismo , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Células-Madre Neurales/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
J Cell Sci ; 125(Pt 22): 5428-40, 2012 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-22946050

RESUMEN

Inositol-(1,4,5)-triphosphate receptors (InsP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores and play a central role in a wide range of cellular responses. In most epithelial cells, InsP(3)Rs are not uniformly distributed within the endoplasmic reticulum (ER) membrane, with the consequence that agonist stimulation results in compartmentalized Ca(2+) signals. Despite these observations, little is known about the mechanisms that regulate the intracellular localization of InsP(3)Rs. Here, we report that exogenously expressed InsP(3)R1-GFP and endogenous InsP(3)R3 interact with the K-Ras-induced actin-binding protein (KRAP) in both differentiated and undifferentiated Madin-Darby canine kidney (MDCK) cells. KRAP mediates InsP(3)R clustering in confluent MDCK cells and functions as an adapter, linking InsP(3)Rs to vimentin intermediate filaments. Upon epithelial differentiation, KRAP and vimentin are both required for InsP(3)R accumulation at the periphery of MDCK cells. Finally, KRAP associates with vimentin in chicken B lymphocytes and with keratins in a breast cancer cell line devoid of vimentin. Collectively, our data suggest that intermediate filaments in conjunction with KRAP may govern the localization of InsP(3)Rs in a large number of cell types (including epithelial cells) and in various physiological or pathological contexts.


Asunto(s)
Diferenciación Celular , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Microfilamentos/metabolismo , Vimentina/metabolismo , Proteínas ras/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Cromatografía Liquida , Perros , Regulación hacia Abajo/efectos de los fármacos , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Queratinas/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/metabolismo , Proteínas Mutantes/metabolismo , Octoxinol/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Espectrometría de Masas en Tándem
6.
Mol Oncol ; 17(12): 2546-2564, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36727672

RESUMEN

Exportin 1 (XPO1) is the main nuclear export receptor that controls the subcellular trafficking and the functions of major regulatory proteins. XPO1 is overexpressed in various cancers and small inhibitors of nuclear export (SINEs) have been developed to inhibit XPO1. In primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin's lymphoma (cHL), the XPO1 gene may be mutated on one nucleotide and encodes the mutant XPO1E571K . To understand the impact of mutation on protein function, we studied the response of PMBL and cHL cells to selinexor, a SINE, and ibrutinib, an inhibitor of Bruton tyrosine kinase. XPO1 mutation renders lymphoma cells more sensitive to selinexor due to a faster degradation of mutant XPO1 compared to the wild-type. We further showed that a mistrafficking of p65 (RELA) and p52 (NFκB2) transcription factors between the nuclear and cytoplasmic compartments accounts for the response toward ibrutinib. XPO1 mutation may be envisaged as a biomarker of the response of PMBL and cHL cells and other B-cell hemopathies to SINEs and drugs that target even indirectly the NFκB signaling pathway.


Asunto(s)
Enfermedad de Hodgkin , Humanos , Línea Celular Tumoral , Proteína Exportina 1 , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Citoplasma/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo
7.
Cell Rep ; 42(9): 113132, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37708024

RESUMEN

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.


Asunto(s)
Melanoma , Multiómica , Humanos , Melanoma/patología , Melanocitos/metabolismo , ADN , Antígenos de Neoplasias/genética
8.
Nat Commun ; 12(1): 5578, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34552068

RESUMEN

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.


Asunto(s)
Células Fotorreceptoras Retinianas Conos/patología , Células Ganglionares de la Retina/metabolismo , Neoplasias de la Retina/clasificación , Retinoblastoma/clasificación , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Desdiferenciación Celular/genética , Preescolar , Metilación de ADN , Femenino , Expresión Génica , Heterogeneidad Genética , Humanos , Lactante , Masculino , Mutación , Proteína Proto-Oncogénica N-Myc/genética , Metástasis de la Neoplasia , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Ganglionares de la Retina/patología , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patología
9.
Exp Eye Res ; 90(6): 791-801, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20380833

RESUMEN

The aim of this work was to define the role of pRb depletion in the proliferation and differentiation of avian retinoblasts in vitro. For this purpose vectors expressing pRb short hairpin RNA were used to deplete pRb in cultures of avian neuroretinal cells. Down regulation of pRb was observed by Western blot and quantification of nuclear pRb. Cell proliferation and differentiation were studied following BrdU labeling and immunostaining. Transfection significantly down-regulated pRb in neuroretinal cells. Long-term effect of pRb depletion mainly induced proliferation of epithelial-like cells that expressed markers of reactive Müller glial cells. A minority of these cells that survived passaging could be maintained as neurosphere-like aggregates with low pRb, not observed in control cultures. BrdU labeling followed by a two week chase showed the presence of cells still remained labelled, indicating low cell cycling. Under appropriate conditions, these aggregates differentiate in precursors of amacrine interneurons shown by the expression of AP2, in absence of the photoreceptors marker visinin and the late neuronal marker MAP2. Taken together these data show that decrease pRb level in cultures of avian neuroretinal cells promotes the emergence and proliferation of stem cell/progenitors from reactive-like Muller cells.


Asunto(s)
Proliferación Celular , Regulación hacia Abajo/fisiología , Neuroglía/citología , Neuronas Retinianas/citología , Proteína de Retinoblastoma/fisiología , Células Madre/citología , Animales , Biomarcadores/metabolismo , Western Blotting , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Embrión de Pollo , Técnica del Anticuerpo Fluorescente Indirecta , Silenciador del Gen , Vectores Genéticos , Hibridación in Situ , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Neuronas Retinianas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Transfección , Vimentina/metabolismo
10.
Genetics ; 179(3): 1345-55, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18562673

RESUMEN

In this study we extend the mouse Pax6 mutant allelic series to include a homozygous and hemizygous viable hypomorph allele. The Pax6(132-14Neu) allele is a Phe272Ile missense mutation within the third helix of the homeodomain. The mutant Pax6 homeodomain shows greatly reduced binding activity to the P3 DNA binding target. Glucagon-promoter activation by the entire mutant Pax6 product of a reporter gene driven by the G1 paired and homeodomain DNA binding target was slightly increased. We constructed mutant Pax6 genotypes such that Pax6 activity ranged between 100 and 0% and show that the extent of eye development is progressively reduced as Pax6 activity decreased. Two apparent thresholds identify three groups in which the extent of eye development abruptly shifted from complete eye at the highest levels of Pax6 to a rudimentary eye at intermediate levels of Pax6 to very early termination of eye development at the lowest levels of Pax6. Of the two Pax6-positive regions that participate in eye development, the surface ectoderm, which develops into the lens vesicle and the cornea, is more sensitive to reduced levels of Pax6 activity than the optic vesicle, which develops into the inner and outer retinal layers.


Asunto(s)
Proteínas del Ojo/metabolismo , Ojo/embriología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Cruzamiento , Mapeo Cromosómico , ADN/metabolismo , Proteínas del Ojo/genética , Femenino , Fertilidad , Regulación del Desarrollo de la Expresión Génica , Genotipo , Glucagón/genética , Heterocigoto , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Mutantes , Tamaño de los Órganos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/genética
11.
J Mol Biol ; 431(17): 3056-3067, 2019 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-31207239

RESUMEN

PRL-3 belongs to the PRL phosphatase family. Its physiological role remains unclear, but many studies have identified that PRL-3 is a marker of cancer progression and shown it to be associated with metastasis. Evidence implicating PRL-3 in various elements of the metastatic process, such as the cell cycle, survival, angiogenesis, adhesion, cytoskeleton remodeling, EMT, motility and invasion, has been reported. Furthermore, several molecules acting as direct or indirect substrates have been identified. However, this information was obtained in many different studies, and it remains difficult to see the larger picture. We therefore systematically collected the published information together and used it to develop a comprehensive signaling network map. By analyzing this network map, we were able to retrieve the signaling pathways via which PRL-3 governs the key steps of the metastatic process in cancer. In this review, we summarize current knowledge of the role of PRL-3 in cancer and the molecular mechanisms involved. We also provide the web-based open-source PRL-3 signaling network map, for use in further studies.


Asunto(s)
Carcinogénesis/metabolismo , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Apoptosis , Adhesión Celular , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Citoesqueleto , Progresión de la Enfermedad , Humanos , Transducción de Señal , Biología de Sistemas
12.
Sci Rep ; 9(1): 2990, 2019 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-30816227

RESUMEN

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Neoplasias de la Úvea/metabolismo , Citoesqueleto de Actina/metabolismo , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Movimiento Celular , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mutación con Pérdida de Función , Melanoma/genética , Melanoma/patología , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Fosfatasas/genética , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patología
13.
Methods Mol Biol ; 1749: 71-77, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29525992

RESUMEN

Metastases is largely responsible for the mortality among cancer patients. Metastasis formation is a complex multistep process, which results from the propagation of cancer cells from the primary tumor to distant sites of the body. Research on cancer metastasis aims to understand the mechanisms involved in the spread of cancer cells through the development of in vivo assays that assess cell invasion. Here we describe the use of the chick chorioallantoic membrane to evaluate cancer cell invasiveness in vivo. The chick chorioallantoic membrane assay is based on the detection and quantification of disseminated human tumor cells in the chick embryo femurs by real-time PCR amplification of human Alu sequences.


Asunto(s)
Movimiento Celular/fisiología , Membrana Corioalantoides/citología , Animales , Línea Celular Tumoral , Embrión de Pollo , Humanos , Metástasis de la Neoplasia , Reacción en Cadena en Tiempo Real de la Polimerasa
14.
Invest Ophthalmol Vis Sci ; 48(12): 5408-19, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18055787

RESUMEN

PURPOSE: Haploinsufficiency and overexpression of the Pax6 gene are responsible for defective central nervous system development. The purpose of the current work was to identify and characterize a new potential role for the Pax6 transcription factor in cellular proliferation in addition to its role at the level of gene expression. METHODS: Expression vectors encoding tagged Pax6p46 protein were used to observe directly protein localization during the cell cycle in cells lines. Three dimensional (3-D) fluorescence microscopy imaging was used to observe in vivo mitotic progression and chromosome dynamics to define the mitotic step affected by p46DsRed as well as to validate endogenous p46 localization on chromosomes in quail retinal cells. Video imaging was used to identify the precise moment of onset of effects related to p46 overexpression in living cells. A pulldown assay in HEK cells was used to identify a specific partner of p46. RESULTS: Pax6p46 protein in transfected cells is localized on the chromosomes, predominantly in a pericentromeric area, and its localization changes as mitosis progresses. Overexpression of p46 protein induces incomplete chromatid separation, resulting in defective mitosis at the onset of the anaphase. A physical interaction between p46 and ESPL1 was identified. CONCLUSIONS: The results suggest that Pax6 exerts an effect on mitosis through protein-protein interactions with proteins localized on chromosomes. Supported by the observation that p46 interacts with separase, an enzyme required for chromatid separation, the authors propose that this interaction is responsible for the mitosis defect observed in cells overexpressing Pax6.


Asunto(s)
Centrosoma/metabolismo , Cromosomas Humanos/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Mitosis/fisiología , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Células Epiteliales , Proteínas del Ojo/genética , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Imagenología Tridimensional , Riñón/citología , Microscopía Fluorescente , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Plásmidos , Codorniz , Proteínas Represoras/genética , Transfección
15.
Invest Ophthalmol Vis Sci ; 57(4): 1982-90, 2016 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-27096756

RESUMEN

PURPOSE: To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness. METHODS: Cell membrane localization of matrix metalloprotease 14 (MMP14) in uveal melanoma cells expressing protein tyrosine phosphatase A3 (PTP4A3) was assessed by flow cytometry or immunohistochemistry. The vesicular trafficking of MMP14 in the presence of PTP4A3 was evaluated in OCM-1 cells expressing either the wild-type or mutated phosphatase. Finally, MMP14 localization at the cell membrane of OCM-1 cells was impaired using RNA interference, and the PTP4A3-related migration in vitro and invasiveness in vivo of the treated cells were evaluated. RESULTS: We found that the membrane-anchored MMP14 is enriched at the cell surface of OCM-1 cells, patient-derived xenograft cells, and human primary uveal melanoma tumors expressing PTP4A3. Moreover, we show that PTP4A3 and MMP14 colocalize and that the vesicular trafficking of MMP14 is faster in the presence of active PTP4A3. Finally, we demonstrate that inhibition of MMP14 expression in uveal melanoma cells expressing PTP4A3 impairs their migration in vitro and invasiveness in vivo. CONCLUSIONS: Our observations indicate that PTP4A3 increases cell membrane accumulation of MMP14 as a result of increased cellular trafficking of the metalloprotease. We also show that downregulation of MMP14 expression reduced PTP4A3-induced cell migration and invasiveness. Taken together, our findings suggest that PTP4A3-related subcellular localization of MMP14 is an important event in metastasis induction.


Asunto(s)
Membrana Celular/metabolismo , Metaloproteinasa 14 de la Matriz/fisiología , Melanoma/fisiopatología , Proteínas de Neoplasias/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Neoplasias de la Úvea/fisiopatología , Línea Celular Tumoral , Movimiento Celular/fisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Melanoma/metabolismo , Invasividad Neoplásica/fisiopatología , Metástasis de la Neoplasia/fisiopatología , Interferencia de ARN , Úvea/metabolismo , Úvea/fisiopatología , Neoplasias de la Úvea/metabolismo
16.
Cell Cycle ; 14(12): 1961-72, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26017556

RESUMEN

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a major role in DNA damage signaling and repair and is also frequently overexpressed in tumor metastasis. We used isogenic cell lines expressing different levels of DNA-PKcs to investigate the role of DNA-PKcs in metastatic development. We found that DNA-PKcs participates in melanoma primary tumor and metastasis development by stimulating angiogenesis, migration and invasion. Comparison of conditioned medium content from DNA-PKcs-proficient and deficient cells reveals that DNA-PKcs controls secretion of at least 103 proteins (including 44 metastasis-associated with FBLN1, SERPINA3, MMP-8, HSPG2 and the inhibitors of matrix metalloproteinases, such as α-2M and TIMP-2). High throughput analysis of secretomes, proteomes and transcriptomes, indicate that DNA-PKcs regulates the secretion of 85 proteins without affecting their gene expression. Our data demonstrate that DNA-PKcs has a pro-metastatic activity via the modification of the tumor microenvironment. This study shows for the first time a direct link between DNA damage repair and cancer metastasis and highlights the importance of DNA-PKcs as a potential target for anti-metastatic treatment.


Asunto(s)
Proteína Quinasa Activada por ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Proteínas Nucleares/fisiología , Animales , Células CHO , Movimiento Celular , Proliferación Celular , Cricetinae , Cricetulus , Medios de Cultivo Condicionados , Daño del ADN , Silenciador del Gen , Humanos , Ganglios Linfáticos/patología , Melanoma/patología , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/metabolismo , Espectrometría de Masas en Tándem
17.
Melanoma Res ; 13(2): 161-6, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12690299

RESUMEN

The KIT gene encodes c-kit, a transmembrane receptor that has tyrosine kinase activity and plays a role in haematopoiesis, gametogenesis and melanogenesis. The c-kit protein is found in normal cutaneous and choroidal melanocytes, and there is evidence that expression is lost in melanoma. Expression of c-kit was analysed in 57 paraffin-embedded sections of choroidal melanoma specimens and three choroidal melanoma cell lines using immunochemistry and Western blotting. Of the tumour specimens, 75% stained positively for c-kit with a membrane pattern of reactivity. Of the six patients who underwent proton beam therapy before enucleation, five tumours exhibited no c-kit immunoreactivity and the other tumour demonstrated weak staining. Of the three melanoma cell lines used, c-kit expression was observed in only one. No correlations between c-kit positivity and parameters such as cell type, largest macroscopic tumour dimension, scleral invasion or pigmentation were observed. In contrast, a significant positive association was found between c-kit staining and mitotic activity (P = 0.02). However, c-kit expression did not significantly influence survival when evaluated by univariate analysis. In conclusion, c-kit is expressed in most choroidal melanoma tumours. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes in choroidal melanomas.


Asunto(s)
Neoplasias de la Coroides/metabolismo , Melanoma/metabolismo , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Mitosis , Factores de Tiempo
18.
PLoS One ; 8(12): e84717, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24376839

RESUMEN

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.


Asunto(s)
Movimiento Celular/fisiología , Metástasis de la Neoplasia/fisiopatología , Cresta Neural/embriología , Proteínas Tirosina Fosfatasas/metabolismo , Cráneo/embriología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Cartilla de ADN/genética , Humanos , Hibridación in Situ , Melanoma/fisiopatología , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Fosfatasas/genética , Cráneo/citología , Imagen de Lapso de Tiempo , Neoplasias de la Úvea/fisiopatología
19.
Mol Oncol ; 7(3): 625-36, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23478236

RESUMEN

We have previously developed a new method for the development and maintenance of uveal melanoma (UM) xenografts in immunodeficient mice. Here, we compare the genetic profiles of the primary tumors to their corresponding xenografts that have been passaged over time. The study included sixteen primary UMs and corresponding xenografts at very early (P1), early (P4), and late (P9) in vivo passages. The tumors were analyzed for mutation status of GNAQ, GNA11, GNAS, GNA15, BAP1, and BRAF, chromosomal copy number alterations using Affymetrix GeneChip(®) Genome-Wide Human SNP6.0 arrays, gene expression profiles using GeneChip(®) Human Exon 1.0 ST arrays, BAP1 mRNA and protein expression, and MAPK pathway status using Reverse Phase Protein Arrays (RPPA). The UM xenografts accurately recapitulated the genetic features of primary human UMs and they exhibited genetic stability over the course of their in vivo maintenance. Our technique for establishing and maintaining primary UMs as xenograft tumors in immunodeficient mice exhibit a high degree of genetic conservation between the primary tumors and the xenograft tumors over multiple passages in vivo. These models therefore constitute valuable preclinical tool for drug screening in UM.


Asunto(s)
Análisis Mutacional de ADN , Melanoma/genética , Transcriptoma , Neoplasias de la Úvea/genética , Animales , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Regulación Neoplásica de la Expresión Génica , Xenoinjertos/metabolismo , Xenoinjertos/patología , Humanos , Sistema de Señalización de MAP Quinasas , Melanoma/metabolismo , Melanoma/patología , Ratones , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas B-raf/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patología
20.
Dev Ophthalmol ; 49: 150-165, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22042019

RESUMEN

Uveal melanoma (UM) arises from neural crest-derived melanocytes of the choroid and the ciliary body. About 50% of patients develop metastatic disease despite efficient control of the primary tumor. For about 15 years, cytogenetic and, recently, genome-wide analysis techniques have shown that UM can be classified into 2 genomic groups correlating with prognostic clinicopathologic features: class 1 tumors, with a low risk of metastases, typically characterized by a gain of the 6p chromosome arm, often associated with a gain of the distal part of the 8q chromosome arm, and class 2 tumors, with a high metastatic risk, presenting loss of the entire chromosome 3 and gain of the entire 8q, related to the formation of isochromosomes. Genome-wide expression profiling has proved to be a powerful tool for separating these 2 classes. However, despite advances in the genomic and prognostic characterization of UM, the knowledge of pathways deregulated in these tumors is just emerging and, in contrast to cutaneous melanoma, no major predisposing genes are known. Altered or deregulated genes are reviewed in this chapter. Inactivating mutations have recently been identified by exome sequencing in gene BAP1, mapping to 3p21.1, in class 2 tumors. Among other discriminant genes identified from genome-wide expression profiling, PTP4A3, mapping to 8q24.3, coding for a protein promoting cell migration, is highly overexpressed in class 2 tumors. The overall expression signature of class 2 tumors suggests they may originate from neuroectodermal stem cells.


Asunto(s)
Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Melanoma/genética , Neoplasias de la Úvea/genética , Biomarcadores de Tumor/biosíntesis , Humanos , Melanoma/metabolismo , Neoplasias de la Úvea/metabolismo
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