RESUMEN
BNIP3 is an atypical BH3-only member of the BCL-2 family of proteins with reported pro-death as well as pro-autophagic and cytoprotective functions, depending on the type of stress and cellular context. In line with this, the role of BNIP3 in cancer is highly controversial and increased BNIP3 levels in cancer patients have been linked with both good as well as poor prognosis. In this study, using small hairpin RNA (shRNA) lentiviral transduction to stably knockdown BNIP3 (BNIP3-shRNA) expression levels in melanoma cells, we show that BNIP3 supports cancer cell survival and long-term clonogenic growth. Although BNIP3-shRNA increased mitochondrial mass and baseline levels of reactive oxygen species production, which are features associated with aggressive cancer cell behavior, it also prevented cell migration and completely abolished the ability to form a tubular-like network on matrigel, a hallmark of vasculogenic mimicry (VM). We found that this attenuated aggressive behavior of these melanoma cells was underscored by severe changes in cell morphology and remodeling of the actin cytoskeleton associated with loss of BNIP3. Indeed, BNIP3-silenced melanoma cells displayed enhanced formation of actin stress fibers and membrane ruffles, while lamellopodial protrusions and filopodia, tight junctions and adherens junctions were reduced. Moreover, loss of BNIP3 resulted in re-organization of focal adhesion sites associated with increased levels of phosphorylated focal adhesion kinase. Remarkably, BNIP3 silencing led to a drop of the protein levels of the integrin-associated protein CD47 and its downstream signaling effectors Rac1 and Cdc42. These observations underscore that BNIP3 is required to maintain steady-state levels of intracellular complexes orchestrating the plasticity of the actin cytoskeleton, which is integral to cell migration and other vital processes stimulating cancer progression. All together these results unveil an unprecedented pro-tumorigenic role of BNIP3 driving melanoma cell's aggressive features, like migration and VM.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Movimiento Celular , Forma de la Célula , Melanoma Experimental/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Citoesqueleto de Actina/patología , Animales , Antígeno CD47/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Adhesiones Focales/metabolismo , Adhesiones Focales/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Proteínas de la Membrana/genética , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Invasividad Neoplásica , Neuropéptidos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
A collection of 106 Photobacterium damselae subsp. piscicida strains and 19 Photobacterium damselae subsp. damselae strains, including reference and type strains, were genetically characterized using AFLP. The total genomic DNA of each bacterial strain was digested using restriction endonucleases HindIII and TaqI. Using numerical analysis, six clusters were recognized. The largest cluster (n = 106) contained the majority of the strains tested and consisted exclusively of Photobacterium damselae subsp. piscicida. The Photobacterium damselae subsp. damselae strains fell outside this cluster. DNA-DNA hybridization experiments showed 77% DNA binding between the two subspecies, indicating a close genetic relationship. This clearly demonstrates the applicability of AFLP in studying the taxonomic position of Photobacterium damselae subsp. piscicida. In addition, AFLP proved to be a useful genotypic technique for epidemiological surveys of the pathogen, since it was able to discriminate between Mediterranean and Japanese Photobacterium damselae subsp. piscicida isolates.
