RESUMEN
Rab proteins are a group of ubiquitously expressed proteins that are responsible for intracellular transport of vesicles. Recent evidence has shown that certain Rab proteins are involved in the pathogenesis of cancer. We have recently shown that Rab25 is lost in a large fraction of breast cancer samples, particularly those derived from hormonally insensitive tumors. We have further investigated the role of Rab25 by re-expressing Rab25 in tumorigenic cell lines and measuring the impact on tumor formation as well as on various molecular pathways through PCR array analysis. In vivo tumor growth of cell lines with re-expressed Rab25 was markedly suppressed. Our data suggest that Rab25 acts through multiple pathways to enhance apoptosis and to suppress angiogenesis and invasion by modulating VEGF-A and VEGFR-1 expression. These findings suggest that Rab25 represents a novel class of cellular modulators that can influence both tumor initiation and the progression of the established tumors, thus ultimately affecting the biology of the malignant disease.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Supresoras de Tumor/fisiología , Proteínas de Unión al GTP rab/fisiología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Adhesión Celular , Ciclo Celular , Movimiento Celular , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Genes Supresores de Tumor , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Ratones , Ratones Desnudos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The growth-retarded (grt) mouse has an autosomal recessive, fetal-onset, severe thyroid hypoplasia related to TSH hyporesponsiveness. Through genetic mapping and complementation experiments, we show that grt is a missense mutation of a highly conserved region of the tyrosylprotein sulfotransferase 2 (Tpst2) gene, encoding one of the two Tpst genes implicated in posttranslational tyrosine O-sulfation. We present evidence that the grt mutation leads to a loss of TPST2 activity, and TPST2 isoform has a high degree of substrate preference for TSH receptor (TSHR). The expression of TPST2 can restore TSH-TSHR-mediated cAMP production in fibroblasts derived from grt mice. Therefore, we propose that the tyrosine sulfation of TSHR by TPST2 is crucial for TSH signaling and resultant thyroid gland function.
Asunto(s)
Hipotiroidismo Congénito/enzimología , Hipotiroidismo Congénito/genética , Enanismo/enzimología , Enanismo/genética , Mutación Missense , Sulfotransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hipotiroidismo Congénito/complicaciones , Cartilla de ADN/genética , Enanismo/complicaciones , Femenino , Prueba de Complementación Genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Fenotipo , Procesamiento Proteico-Postraduccional , Receptores de Tirotropina/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por Sustrato , Sulfotransferasas/deficiencia , Sulfotransferasas/metabolismo , Tirotropina/metabolismoRESUMEN
Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (approximately 40 kDa) bound to DEAE-Sepharose column and protein complexes (approximately 300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by co-immunoprecipitation with ACCA antibody and pulldown assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, small interfering RNA-mediated AKR1B10 knock down increased ACCA degradation through ubiquitination-proteasome pathway and resulted in >50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells.
Asunto(s)
Acetil-CoA Carboxilasa/química , Aldehído Reductasa/fisiología , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Acetil-CoA Carboxilasa/metabolismo , Aldehído Reductasa/genética , Aldo-Ceto Reductasas , Línea Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , Ácidos Grasos/metabolismo , Silenciador del Gen , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ubiquitina/metabolismoRESUMEN
A novel breast cancer cell line (RAO-3) was established by transduction of the Q61L mutant RAS into human mammary epithelial cells that were immortalized with catalytic subunit of telomerase (hTERT). The cells displayed anchorage-independent growth and proliferation, and formed human mammary spindle cell carcinoma when injected into nude mice. Chromosome locus 1q22-23 was partially duplicated and inverted on one of the 3 chromosomes present in the cell line. We report here that mutations of chromosome 1q22-23 locus have resulted in the loss of RAB25 expression in the breast cancer cell line. Transduction of RAB25 into the breast cancer cell line arrests anchorage-independent growth. We have also demonstrated loss of RAB25 in human breast tumor tissue. These data suggest that loss of RAB25 might contribute to tumorigenesis of breast cancer, and RAB25 is likely to be an important factor in the development of breast cancer. RAB25 could be used as biological marker of breast cancer and provides a target for gene replacement therapy.