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.

Figure 1

BNIP3 is required to maintain clonogenic growth of melanoma cells. (a) Confirmation of effective BNIP3 KD in control and BNIP3 shRNA transduced B16-F10 cells on protein level (24 h after cell plating). A representative western blot (n=6) probed with anti-BNIP3 antibody and quantification below is shown. Actin is used as a loading control. Statistical analysis was performed as described in the materials and methods section. (b) The effect of BNIP3 silencing on spontaneous cell death determined by PI exclusion. Graph shows the percentage of dead cells (PI-positive) of control and BNIP3 KD conditions as a function of time after B16-F10 cell plating (12, 24 and 48 h) (n=3). (c) Representative images (n=3) showing clonogenic expansion of control and BNIP3 silenced B16-F10 cells. Photos were taken 10 days after seeding of the cells as a diluted single cell suspension

Figure 2

BNIP3 modulates autophagic degradation of mitochondria and ROS signaling in melanoma. (a) The effect of BNIP3 KD on basal cellular LC3I to LC3II conversion in B16-F10 cells was determined by western blot (24 h after cell plating). A representative western blot (n=7) probed with anti-LC3 antibody of control and BNIP3 KD conditions and quantifications are shown. Actin is used as a loading control. (b) Assessment of the effect of BNIP3 KD on cellular ROS levels in B16-F10 cells (24 h after cell plating). Cellular fluorescence (green) is shown for control and BNIP3 KD conditions after incubation of the cells with CM-H2DCFDA (10 μM) for 30 min (n=6). (c) Analysis of the mitochondrial DNA content of control and BNIP3 KD B16-F10 cells assessed as the ratio of mitochondrial (LPL)/ nuclear (ND1) DNA copy numbers (n=5). (d) Assessment of the effect of BNIP3 KD on cellular Mitotracker uptake in B16-F10 cells (24 h after cell plating). Cellular fluorescence (deep red) is shown for control and BNIP3 KD conditions after incubation of the cells with Mitotracker (25 nM) for 30 min (n=3) and (e) representative confocal images (n=3, five pictures per condition) of control and BNIP3 KD B16-F10 cells (24 h after cell plating), after immunostaining for TOMM20 and the nuclear dye DAPI, scale bars represent 10 μM

Figure 3

BNIP3 promotes melanoma cell migration and VM. (a) Two-dimensional cell migration of control versus BNIP3 KD B16-F10 cells. Representative images (n=3) of the scratch at indicated time points and quantification of the wound area (size wound at Tx/size wound at T0) are shown, arrows indicate the wound area, scale bars represent 400 μm. (b) The ability of the B16-F10 cells to form a vascular-like network was assessed by plating the cells on growth factor reduced Matrigel. Representative images (n=4) 16 h after cell seeding and quantification are shown of control and BNIP3 KD cells, HUVEC were used as a positive control, scale bars represent 200 μm

Figure 4

BNIP3 is required to maintain cellular architecture and cytoskeletal structures. (a) Representative light microscopy images (n=4, five pictures per experiment) of control and BNIP3 KD B16-F10 cells (24 h after cell plating), scale bars represent 50 μm. (b) Cell surface area, quantified using ImageJ, of control and BNIP3 KD cells. (c) Nuclear size, quantified using Image J, of control and BNIP3 KD cells. (d) Confocal microscopy analysis of the actin cytoskeleton of B16-F10 cells (24 h after cell plating) using the high affinity actin-probe phalloidin-Alexa Flour 488 and the nuclear dye DAPI. Representative images (n=5, five pictures per condition) of control versus BNIP3 KD cells, scale bars represent 10 μm. (e) Confocal microscopy analysis of the actin cytoskeleton using the high affinity actin-probe Phalloidin-Alexa Flour 488 and the nuclear dye DAPI on low density cell culture of B16-F10 cells (24 h after cell plating). Representative images (n=5, five pictures per condition) of control versus BNIP3 KD cells are shown. Scale bars represent 10 μm. Arrows indicate lamellipodia and arrowheads indicate membrane ruffles. (f) Higher magnification images of the staining in e to reveal cell filopodia. Representative images (n=5, five pictures per condition) and zoom of control and BNIP3 KD B16-F10 cells are shown, scale bar represents 10 μm, arrows indicate filopodia

Figure 5

BNIP3 is essential to maintain balanced FAs. (a–c) Analysis of the protein levels of integrin (a), P-FAK (b) and vinculin (c) of B16-F10 cells (24 h after cell plating) using immunoblotting, a representative western blot (n=3) and quantifications of control and BNIP3 KD samples is shown, actin is used to verify equal loading. (d) Representative confocal images (n=3, five pictures per condition) of control and BNIP3 KD B16-F10 cells (24 h after cell plating), after immunostaining for integrin αv and incubation with Phalloidin-Rhodamine, scale bars represent 10 μM. (e) Representative confocal images (n=3, five pictures per condition) of control and BNIP3 KD B16-F10 cells (24 h after cell plating), after immunostaining for P-FAK and incubation with Phalloidin-Rhodamine, scale bars represent 10 μM. (f) Representative confocal images (n=3, five pictures per condition) of control and BNIP3 KD cells (24 h after cell plating), after immunostaining for vinculin and incubation with Phalloidin-Rhodamine, scale bars represent 10 μM

Figure 6

BNIP3 modulates Rac1 and Cdc42, key signaling molecules in the VM phenotype. (a) Analysis of the protein levels of Rac1 and Cdc42 in control and BNIP3 KD B16-F10 cells (24 h after cell plating). A representative immunoblot probed with anti-Rac1 (n=4) or Cdc42 (n=3) antibody is shown, actin is used to verify equal loading. (b) Analysis of the activation status of Rac1 and Cdc42 of B16-F10 cells (18 h after cell plating) after pull down of their GTP-bound forms using PAK-coated beads. Representative western blots of the bead-bound fraction and total cell lysate (not corrected for protein concentration) probed with Rac1 antibody (n=4) or Cdc42 antibody (n=3) are shown. (c) Two-dimensional cell migration of control versus Rac inhibitor (EHop-016; 1 μM-24 h) and/or Cdc42 inhibitor (ML-141; 2 μM-24 h) treated cells. Representative images (n=3) of the scratch wound at time point 0 h, 18 h and zoom of 18 h and quantification of the wound area (size wound at Tx/size wound at T0) are shown. Arrows indicate the wound area, scale bars represent 800 μm. (d) The ability of the B16-F10 cells to form a vascular-like network was assessed by plating the cells on growth factor reduced Matrigel (16 h after cell plating). Representative images (n=4) and quantification are shown of control versus Rac inhibitor (EHop-016; 1 μM-16 h) and/or Cdc42 inhibitor (ML-141; 2 μM-16 h) treated cells, scale bars represent 200 μm

Figure 7

BNIP3 KD reduces the protein levels of CD47, associated with reduced cell–cell interaction. (a) Analysis of the effect of BNIP3 KD B16-F10 cells on CD47 protein level (24 h after cell plating). A representative western blot (n=6) probed with anti-CD47 antibody of control and BNIP3 KD cells is shown. Actin is used as a loading control. (b) Analysis of the effect of hypoxia (1.5%O₂-24 h)-induced BNIP3 on CD47 protein level as compared with BNIP3 KD (24 h after cell plating). A representative western blot (n=3) probed with anti-CD47 antibody of EV normoxia, EV hypoxia and BNIP3 KD B16-F10 cells (normoxia) is shown, actin is used to verify equal loading. Note that the baseline levels of BNIP3 in this western blot are almost not visible due to lower exposure chosen to avoid overexposure of the signal of the hypoxia-mediated BNIP3 induction. (c) Immunoblotting for the adherens junction protein VE–cadherin and the tight junction protein ZO-1 (24 h after cell plating) of B16-F10 cells, actin is used to verify equal loading (n=3). (d) Confocal microscopy analysis after immunostaining for ZO-1 to reveal tight junctions and Phalloidin-Alexa 488 to document cellular orientation combined with the nuclear dye DAPI of B16-F10 cells (24 h after cell plating). Representative pictures (n=3) of control and BNIP3 KD cells are shown, scale bar represents 10 μM. (e) Effect of proteasome inhibition via MG132 10 μM-3 h (MG132) or inhibition of lysosomal degradation via Bafilomycin A1 10 nM-3 h (BAF) on CD47 protein levels in control and BNIP3 KD B16-F10 cells. Quantification of immunoblots (n=4) probed with anti-CD47 antibody of control and BNIP3 KD cells treated with DMSO, LAC and BAF (relative to DMSO-treated control conditions) are shown, actin is used to verify equal loading. (f) A scatterplot of correlation between CD47 transcript expression levels and BNIP3 transcript expression levels in a cohort of skin cutaneous melanoma patients (n=312). Correlation levels have been estimated on the basis of correlation coefficient (Corr. Coef.) and the P-value for this correlation (P_Corr), both of which are displayed on the plot. Here, a Corr. Coef.=1 represents a perfect correlation between the variables while Corr. Coef. between 0 and 1 denotes that the two variables tend to increase or decrease together