title: BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton.
abstract: 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.
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: H
authors.initials: H
authors.lastname: Maes
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Van Eygen
authors.affiliation: 1] Molecular signaling and Cell Death Unit, Inflammation Research Center, VIB, Ghent, Belgium [2] Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: D V
authors.initials: DV
authors.lastname: Krysko
authors.affiliation: 1] Molecular signaling and Cell Death Unit, Inflammation Research Center, VIB, Ghent, Belgium [2] Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: P
authors.initials: P
authors.lastname: Vandenabeele
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Nys
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Rillaerts
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: A D
authors.identifier: 0000000299769922
authors.initials: AD
authors.lastname: Garg
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: T
authors.identifier: 0000000158855376
authors.initials: T
authors.lastname: Verfaillie
authors.affiliation: Laboratory of Cell Death Research and Therapy, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: P
authors.initials: P
authors.lastname: Agostinis
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D051928:CD47 Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D048430:Cell Shape; D002470:Cell Survival; D022001:Focal Adhesions / Q000378:metabolism / Q000473:pathology; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009479:Neuropeptides / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: C090121:BNIP3 protein, human; C508686:BNip3 protein, mouse; D051928:CD47 Antigen; C497895:Cd47 protein, mouse; C581598:Cdc42 protein, mouse; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009479:Neuropeptides; D011518:Proto-Oncogene Proteins; C484602:Rac1 protein, mouse; D017382:Reactive Oxygen Species; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein