RESUMEN
Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.
Asunto(s)
Galectina 1/metabolismo , Mieloma Múltiple/patología , Neovascularización Patológica/tratamiento farmacológico , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular , Galectina 1/antagonistas & inhibidores , Humanos , Ratones , Mieloma Múltiple/irrigación sanguínea , ARN Interferente Pequeño/farmacología , Transfección , Carga Tumoral/efectos de los fármacosAsunto(s)
Activinas/fisiología , Enfermedades Óseas/etiología , Interleucina-3/farmacología , Macrófagos/fisiología , Monocitos/fisiología , Mieloma Múltiple/complicaciones , Osteoclastos/fisiología , Osteogénesis/efectos de los fármacos , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Ligando RANK/fisiologíaRESUMEN
Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Neovascularización Patológica/genética , Osteólisis/genética , Osteólisis/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Carga Tumoral/genéticaRESUMEN
Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Canonical Wnt signaling is critical for the regulation of bone formation, however, recent evidence suggests that the non-canonical Wnt agonist Wnt5a stimulates human osteoblastogenesis through its co-receptor Ror2. The effects of MM cells on non-canonical Wnt signaling and the effect of the activation of this pathway on MM-induced osteoblast exhaustion are not known and were investigated in this study. We found that the osteogenic differentiation of bone marrow hMSCs toward osteoprogenitor cells (PreOB) significantly increased Ror2 expression, and that MM cells inhibit Ror2 expression by PreOB in co-culture by inhibiting the non-canonical Wnt5a signaling. The activation of the non-canonical Wnt pathway in hMSCs by means of Wnt5a treatment and the overexpression of Wnt5 or Ror2 by lentiviral vectors increased the osteogenic differentiation of hMSCs and blunted the inhibitory effect of MM in co-culture. Consistently, Wnt5a inhibition by specific small interfering RNA reduced the hMSC expression of osteogenic markers. Our findings demonstrate that the Wnt5a/Ror2 pathway is involved in the pathophysiology of MM-induced bone disease and that the activation of the non-canonical Wnt5a/Ror2 pathway in hMSCs increases osteogenic differentiation and may counterbalance the inhibitory effect of MM cells.
Asunto(s)
Células de la Médula Ósea/citología , Diferenciación Celular , Mieloma Múltiple/patología , Osteoblastos/citología , Osteogénesis , Proteínas Proto-Oncogénicas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Células Madre/citología , Proteínas Wnt/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Células de la Médula Ósea/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Técnicas de Cocultivo , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Madre/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
The involvement of osteocytes in multiple myeloma (MM)-induced osteoclast (OCL) formation and bone lesions is still unknown. Osteocytes regulate bone remodelling at least partially, as a result of their cell death triggering OCL recruitment. In this study, we found that the number of viable osteocytes was significantly smaller in MM patients than in healthy controls, and negatively correlated with the number of OCLs. Moreover, the MM patients with bone lesions had a significantly smaller number of viable osteocytes than those without, partly because of increased apoptosis. These findings were further confirmed by ultrastructural in vitro analyses of human preosteocyte cells cocultured with MM cells, which showed that MM cells increased preosteocyte death and apoptosis. A micro-array analysis showed that MM cells affect the transcriptional profiles of preosteocytes by upregulating the production of osteoclastogenic cytokines such as interleukin (IL)-11, and increasing their pro-osteoclastogenic properties. Finally, the osteocyte expression of IL-11 was higher in the MM patients with than in those without bone lesions. Our data suggest that MM patients are characterized by a reduced number of viable osteocytes related to the presence of bone lesions, and that this is involved in MM-induced OCL formation.
Asunto(s)
Apoptosis , Biomarcadores de Tumor/genética , Interleucina-11/genética , Gammopatía Monoclonal de Relevancia Indeterminada/patología , Mieloma Múltiple/patología , Osteoclastos/patología , Osteocitos/patología , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Western Blotting , Resorción Ósea , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Interleucina-11/metabolismo , Masculino , Persona de Mediana Edad , Gammopatía Monoclonal de Relevancia Indeterminada/genética , Gammopatía Monoclonal de Relevancia Indeterminada/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoclastos/metabolismo , Osteocitos/metabolismo , Osteogénesis/fisiología , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The deregulation of the homeobox genes as homeoboxB (HOXB)-7 has been previously associated to tumor progression and angiogenesis; here we investigated the potential role of HOXB7 in the pro-angiogenic properties of multiple myeloma (MM) cells. We found that HOXB7 was expressed in 10 out of 22 MM patients analyzed at the diagnosis related to high bone marrow angiogenesis and overexpressed in about 40% of myeloma cell lines compared with normal plasma cells. Enforced HOXB7 expression in MM cells by a lentiviral vector significantly modified their transcriptional and angiogenic profile, checked by combined microarray and angiogenesis PCR analyses, upregulating VEGFA, FGF2, MMP2, WNT5a and PDGFA and downregulating thrombospoindin-2. The pro- and anti-angiogenic HOXB7-related gene signature was also validated in a large independent dataset of MM patients. Accordingly, MM-induced vessel formation was significantly increased by HOXB7 overexpression both in vitro angiogenic and chorioallantoic membrane assays, as well as the HOXB7 silencing by small interfering RNA inhibited the production of angiogenic factors, and the pro-angiogenic properties of MM cells. Finally, in SCID-NOD mice we confirmed that HOXB7 overexpression by MM cells stimulated tumor growth, increased MM-associated angiogenesis and the expression of pro-angiogenic genes by microarray analysis supporting the critical role of HOXB7 in the angiogenic switch in MM.
Asunto(s)
Proteínas de Homeodominio/fisiología , Mieloma Múltiple/irrigación sanguínea , Neovascularización Patológica/etiología , Anciano , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Factor A de Crecimiento Endotelial Vascular/biosíntesisAsunto(s)
Neoplasias Óseas/patología , Mesodermo/patología , Mieloma Múltiple/patología , Osteoblastos/patología , Células del Estroma/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Huesos/citología , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mesodermo/metabolismo , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/metabolismo , Células del Estroma/metabolismo , Telómero/genéticaRESUMEN
Wastewater concentrates from the wastewater treatment systems of three dye plants were tested for mutagenic activity in Salmonella typhimurium TA98 and Escherichia coli WP2uvrA using a fluctuation assay. Concentrates were prepared by passing samples of wastewater (5-6 or 30 litres) through two porous resins (XAD-2 and XAD-7) in series. S. typhimurium in the presence of microsomal activation proved to be the more sensitive marker of mutagenicity. Mutagenic responses were observed in concentrates from all three plants tested. The results show that mutagenic activity was particularly high in the incoming waters and increased after active, biological treatment. Physico-chemical treatment may be effective in decreasing mutagenic activity, but only if appropriately used.