RESUMEN
The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial-fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D-deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras-MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.
Asunto(s)
Catepsina D/fisiología , Movimiento Celular/fisiología , Fibroblastos/citología , Animales , Apoptosis/fisiología , Butadienos/farmacología , Catepsina D/genética , Catepsina D/metabolismo , Aumento de la Célula/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , Manosafosfatos/farmacología , Ratones , Microscopía Electrónica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Invasividad Neoplásica , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Nitrilos/farmacología , Comunicación Paracrina/fisiología , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Transfección , Cicatrización de HeridasRESUMEN
Defects in insulin secretion, resulting from loss of function or destruction of pancreatic beta-cells, trigger diabetes. Interleukin (IL)-1beta is a proinflammatory cytokine that is involved in type 1 and type 2 diabetes development and impairs beta-cell survival and function. Because effective insulin signaling is required for the optimal beta-cell function, we assessed the effect of IL-1beta on the insulin pathway in a rat pancreatic beta-cell line. We show that IL-1beta decreases insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS) proteins as well as phosphatidylinositol 3-kinase (PI3K) activation, and that this action is not due to the IL-1beta-dependent nitric oxide (NO) production in RINm5F cells. We next analyzed if suppressor of cytokine signaling (SOCS)-3, which can be induced by multiple cytokines and which we identified as an insulin action inhibitor, was implicated in the IL-1beta inhibitory effect on insulin signaling in these cells. We show that IL-1beta increases SOCS-3 expression and induces SOCS-3/IR complex formation in RINm5F cells. Moreover, we find that ectopically expressed SOCS-3 associates with the IR and reduces insulin-dependent IR autophosphorylation and IRS/PI3K pathway in a way comparable to IL-1beta treatment in RINm5F cells. We propose that IL-1beta decreases insulin action in beta-cells through the induction of SOCS-3 expression, and that this effect potentially alters insulin-induced beta-cell survival.
Asunto(s)
Insulina/metabolismo , Interleucina-1/farmacología , Islotes Pancreáticos/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Animales , Línea Celular Tumoral , Clonación Molecular , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Secreción de Insulina , Insulinoma , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Neoplasias Pancreáticas , Fosforilación , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Factores de Transcripción/genéticaRESUMEN
Cathepsin-D is an independent marker of poor prognosis in human breast cancer. We previously showed that human wild-type cathepsin-D, as well as its mutated form devoid of proteolytic activity stably transfected in 3Y1-Ad12 cancer cells, stimulated tumor growth. To investigate the mechanisms by which human cathepsin-D and its catalytically-inactive counterpart promoted tumor growth in vivo, we quantified the expression of proliferating cell nuclear antigen, the number of blood vessels and of apoptotic cells in 3Y1-Ad12 tumor xenografts. We first verified that both human wild-type and mutated cathepsin-D were expressed at a high level in cathepsin-D xenografts, whereas no human cathepsin-D was detected in control xenografts. Our immunohistochemical studies then revealed that both wild-type cathepsin-D and catalytically-inactive cathepsin-D, increased proliferating cell nuclear antigen expression and tumor angiogenesis. Interestingly, wild-type cathepsin-D significantly inhibited tumor apoptosis, whereas catalytically-inactive cathepsin-D did not. We therefore propose that human cathepsin-D stimulates tumor growth by acting-directly or indirectly-as a mitogenic factor on both cancer and endothelial cells independently of its catalytic activity. Our overall results provide the first mechanistic evidences on the essential role of cathepsin-D at multiple tumor progression steps, affecting cell proliferation, angiogenesis and apoptosis.
Asunto(s)
Catepsina D/fisiología , Neovascularización Patológica , Animales , Apoptosis/fisiología , División Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Mutación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Overexpression of cathepsin-D in primary breast cancer has been associated with rapid development of clinical metastasis. To investigate the role of this protease in breast cancer growth and progression to metastasis, we stably transfected a highly metastatic human breast cancer cell line, MDA-MB-231, with a plasmid containing either the full-length cDNA for cathepsin-D or a 535 bp antisense cathepsin-D cDNA fragment. Clones expressing antisense cathepsin-D cDNA that exhibited a 70-80% reduction in cathepsin-D protein, both intra- and extracellularly compared to controls, were selected for further experiments. These antisense-transfected cells displayed a reduced outgrowth rate when embedded in a Matrigel matrix, formed smaller colonies in soft agar and presented a significantly decreased tumor growth and experimental lung metastasis in nude mice compared with controls. However, manipulating the cathepsin-D level in the antisense cells has no effect on their in vitro invasiveness. These studies demonstrate that cathepsin-D enhances anchorage-independent cell proliferation and subsequently facilitates tumorigenesis and metastasis of breast cancer cells. Our overall results provide the first evidence on the essential role of cathepsin-D in breast cancer, and support the development of a new cathepsin-D-targeted therapy.