RESUMEN
Metastatic spread in Ewing sarcomas (ES) is frequent and haematogenous. G-protein coupled receptor 64 (GPR64), an orphan receptor with normal expression restricted to human epididymis is specifically over-expressed in ES among sarcoma, but also up-regulated in a number of carcinomas derived from prostate, kidney or lung. Inhibition of GPR64 expression in ES by RNA interference impaired colony formation in vitro and suppressed local tumour growth and metastasis in Rag2(-/-) γC (-/-) mice. Microarray analysis after GPR64 knock down revealed a GPR64-mediated repression of genes involved in neuronal development like SLIT, drosophila, homolog of, 2 (SLIT2), and genes regulating transcription including pre-B cell leukemia homeobox 2 (PBX2). Concurrently, the suppression of GPR64 increased ES susceptibility to TRAIL induced apoptosis. Moreover, a GPR64-mediated induction of placental growth factor (PGF) in ES was observed. PGF suppression by RNA interference resulted in a reduction of metastatic growth similar to that observed after GPR64 knock down. Importantly, inhibition of GPR64 as well as PGF expression was associated with a reduced expression of matrix metalloproteinase (MMP) 1 and invasiveness in vitro. Furthermore, MMP1 knock down abrogated lung metastasis in Rag2(-/-) γC (-/-) mice. Thus, GPR64 expression in ES maintains an immature phenotype that is less sensitive to TRAIL-induced apoptosis and via its up-regulation of PGF and MMP1 orchestrates and promotes invasiveness and metastatic spread.
Asunto(s)
Neoplasias Óseas/patología , Metaloproteinasa 13 de la Matriz/metabolismo , Proteínas Gestacionales/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sarcoma de Ewing/secundario , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Invasividad Neoplásica/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma , Factor de Crecimiento Placentario , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiologíaRESUMEN
Ewing sarcoma, an osteolytic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. In this study, we identified the pro-metastatic gene DKK2 as a highly overexpressed gene in Ewing sarcoma compared with corresponding normal tissues. Using RNA interference, we showed that DKK2 was critical for malignant cell outgrowth in vitro and in an orthotopic xenograft mouse model in vivo. Analysis of invasion potential in both settings revealed a strong correlation of DKK2 expression to Ewing sarcoma invasiveness that may be mediated by the DKK effector matrix metalloproteinase 1 (MMP1). Furthermore, gene expression analyses established the ability of DKK2 to differentially regulate genes such as CXCR4, PTHrP, RUNX2, and TGFß1 that are associated with homing, invasion, and growth of cancer cells in bone tissue as well as genes important for osteolysis, including HIF1α, JAG1, IL6, and VEGF. DKK2 promoted bone infiltration and osteolysis in vivo and further analyses defined DKK2 as a key factor in osteotropic malignancy. Interestingly, in Ewing sarcoma cells, DKK2 suppression simultaneously increased the potential for neuronal differentiation while decreasing chondrogenic and osteogenic differentiation. Our results provide strong evidence that DKK2 is a key player in Ewing sarcoma invasion and osteolysis and also in the differential phenotype of Ewing sarcoma cells.
Asunto(s)
Neoplasias Óseas/secundario , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Osteólisis , Sarcoma de Ewing/patología , Animales , Diferenciación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Neuronas/citología , Sarcoma de Ewing/metabolismo , Trasplante Heterólogo , Regulación hacia ArribaRESUMEN
Ewing tumors comprise the second most common type of bone-associated cancer in children and are characterized by oncogenic EWS/FLI1 fusion proteins and early metastasis. Compelling evidence suggests that elevated levels of intracellular oxidative stress contribute to enhanced aggressiveness of numerous cancers, possibly including Ewing tumors. Using comprehensive microarray analyses and RNA interference, we identified the six-transmembrane epithelial antigen of the prostate 1 (STEAP1)-a membrane-bound mesenchymal stem cell marker of unknown function-as a highly expressed protein in Ewing tumors compared with benign tissues and show its regulation by EWS/FLI1. In addition, we show that STEAP1 knockdown reduces Ewing tumor proliferation, anchorage-independent colony formation as well as invasion in vitro and decreases growth and metastasis of Ewing tumor xenografts in vivo. Moreover, transcriptome and proteome analyses as well as functional studies revealed that STEAP1 expression correlates with oxidative stress responses and elevated levels of reactive oxygen species that in turn are able to regulate redox-sensitive and proinvasive genes. In synopsis, our data suggest that STEAP1 is associated with the invasive behavior and oxidative stress phenotype of Ewing tumors and point to a hitherto unanticipated oncogenic function of STEAP1.