BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton

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

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression

A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK) and Src, and that this signalling pathway lies downstream of fascin-microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and may have implications for the design of therapies to target fascin in metastatic disease

LUZP1 Controls Cell Division, Migration and Invasion Through Regulation of the Actin Cytoskeleton

LUZP1 is a centrosomal and actin cytoskeleton-localizing protein that regulates both ciliogenesis and actin filament bundling. As the cytoskeleton and cilia are implicated in metastasis and tumor suppression, we examined roles for LUZP1 in the context of cancer. Here we show that LUZP1 exhibits frequent genomic aberrations in cancer, with a predominance of gene deletions. Furthermore, we demonstrate that CRISPR/Cas9-mediated loss of Luzp1 in mouse fibroblasts promotes cell migration and invasion features, reduces cell viability, and increases cell apoptosis, centriole numbers, and nuclear size while altering the actin cytoskeleton. Loss of Luzp1 also induced changes to ACTR3 (Actin Related Protein 3, also known as ARP3) and phospho-cofilin ratios, suggesting regulatory roles in actin polymerization, beyond its role in filament bundling. Our results point to an unprecedented role for LUZP1 in the regulation of cancer features through the control of actin cytoskeleton.We are grateful to the Fundacion Inocente, Inocente for their support. We also acknowledge funding by the grants BFU2017-84653-P (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), 765445-EU (UbiCODE Program), SAF2017-90900-REDT (UBIRed Program), and IT1165-19 (Basque Country Government). Additional support was provided by the Department of Industry, Tourism, and Trade of the Basque Country Government (Elkartek Research Programs) and by the Innovation Technology Department of the Bizkaia County. LB-B acknowledges POSTD19048BOZA (Fundacion Cientifica AECC). VM acknowledges PRE2018086230 (MINECO/FEDER, EU). AC acknowledges the Basque Department of education (IKERTALDE IT1106-16), the MCIU [PID2019-108787RB-I00 (FEDER/EU)], the AECC (IDEAS175CARR; GCTRA18006CARR), La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17/and the European Research Council (Starting Grant 336343, PoC 754627, Consolidator grant 819242). CIBERONC was co-funded with FEDER funds. We are also grateful to Maria Vivanco's lab for providing reagents

Identifying new regulators of the actin cytoskeleton in endothelial tip cells

Blood vessels form extensive vascular networks allowing an efficient transport of gases, fluids and nutrients for all tissues. Sprouting angiogenesis generates new blood vessels during embryonic development and adult. Dysfunctional, either excessive or insufficient, angiogenesis is a cause of several vascular diseases, such as ischemia, arteriovenous malformations, tumor angiogenesis, and diabetic retinopathy. During sprouting angiogenesis, endothelial tip cells are highly migratory and guide the trailing endothelial stalk cells in the nascent sprout towards the source of pro-angiogenic factors. However, the mechanisms regulating the actin cytoskeleton contributing to the motility and invasive proprieties of endothelial tip cells are poorly understood. Serum response factor (SRF) is a transcription factor that regulates expression of genes encoding cytoskeletal proteins. SRF, together with its cofactors [myocardin related transcription factors (MRTFs)], is essential for tip cell migration and invasion during sprouting angiogenesis. But which genes downstream of SRF/MRTF signaling are central in this process are completely not characterized. Based on a microarray approach, we defined the MRTF/SRF-dependent transcriptome of endothelial cells. Using a combination of multiple in silico and in vitro approaches, we identified the relevant genes downstream of SRF signaling, and we characterize their function in cell migration and cytoarchitecture of endothelial cells. From our analysis, inhibition of MYH9, the gene coding for myosin IIA heavy chain (NMII-A), reproduced alone the full spectrum of phenotypes presented in MRTF/SRF-deficient endothelium, and regulates tip cell motility and invasion. Interestingly, we found a specialized localization of MYH9 in endothelial tip cells, pointing towards an important and novel role in filopodia formation and/or stability. Our research provides new insights on the biology of endothelial tip cells, which can be a first step towards new therapeutic approaches targeting pathological vascularization

Effects of Noninhibitory Serpin Maspin on the Actin Cytoskeleton: A Quantitative Image Modeling Approach

Recent developments in quantitative image analysis allow us to interrogate confocal microscopy images to answer biological questions. Clumped and layered cell nuclei and cytoplasm in confocal images challenges the ability to identify subcellular compartments. To date, there is no perfect image analysis method to identify cytoskeletal changes in confocal images. Here, we present a multidisciplinary study where an image analysis model was developed to allow quantitative measurements of changes in the cytoskeleton of cells with different maspin exposure. Maspin, a noninhibitory serpin influences cell migration, adhesion, invasion, proliferation, and apoptosis in ways that are consistent with its identification as a tumor metastasis suppressor. Using different cell types, we tested the hypothesis that reduction in cell migration by maspin would be reflected in the architecture of the actin cytoskeleton. A hybrid marker-controlled watershed segmentation technique was used to segment the nuclei, cytoplasm, and ruffling regions before measuring cytoskeletal changes. This was informed by immunohistochemical staining of cells transfected stably or transiently with maspin proteins, or with added bioactive peptides or protein. Image analysis results showed that the effects of maspin were mirrored by effects on cell architecture, in a way that could be described quantitatively

Src-Dependent Tyrosine Phosphorylation at the Tips of Growth Cone Filopodia Promotes Extension

Extracellular cues guide axon outgrowth by activating intracellular signaling cascades that control the growth cone cytoskeleton. However, the spatial and temporal coordination of signaling intermediates remains essentially unknown. Live imaging of tyrosine phosphorylation in growth cones revealed dynamic phospho-tyrosine (PY) signals in filopodia that directly correlate with filopodial behavior. Local PY signals are generated at distal tips of filopodia during extension and are lost during retraction. Active Src family kinases localize to the tips of filopodia, and Src activity regulates both filopodial dynamics and local PY signaling. Positive guidance cues stimulate filopodial motility by locally increasing tyrosine phosphorylation in a cell division cycle 42 (Cdc42)-dependent manner. Locally reduced Src activity on one side of the growth cone generates an asymmetry in filopodial motility and PY signaling that promotes repulsive turning, suggesting that local changes in filopodial PY levels may underlie growth cone pathfinding decisions. p21-activated kinase (PAK), a Cdc42 effector whose activity is regulated by Src phosphorylation, also localizes to the tips of extending filopodia and controls filopodial motility. Coordinated activation of cytoskeletal effector proteins by GTPase binding and Src-mediated tyrosine phosphorylation may function to produce specific growth cone behaviors in response to guidance cues

Spatiotemporal endothelial cell-pericyte association in tumors as shown by high resolution 4D intravital imaging

Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell-pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells