Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

<div><p>Bcl-w a pro-survival member of the Bcl-2 protein family, is expressed in a variety of cancer types, including gastric and colorectal adenocarcinomas, as well as glioblastoma multiforme (GBM), the most common and lethal brain tumor type. Previously, we demonstrated that Bcl-w is upregulated in gastric cancer cells, particularly those displaying infiltrative morphology. These reports propose that Bcl-w is strongly associated with aggressive characteristic, such as invasive or mesenchymal phenotype of GBM. However, there <i>is no information</i> from studies of the role of Bcl-w in GBM. In the current study, we showed that Bcl-w is upregulated in human glioblastoma multiforme (WHO grade IV) tissues, compared with normal and glioma (WHO grade III) tissues. Bcl-w promotes the mesenchymal traits of glioblastoma cells by inducing vimentin expression via activation of transcription factors, β-catenin, Twist1 and Snail in glioblastoma U251 cells. Moreover, Bcl-w induces invasiveness by promoting MMP-2 and FAK activation via the PI3K-p-Akt-p-GSK3β-β-catenin pathway. We further confirmed that Bcl-w has the capacity to induce invasiveness in several human cancer cell lines. In particular, Bcl-w-stimulated β-catenin is translocated into the nucleus as a transcription factor and promotes the expression of target genes, such as mesenchymal markers or MMPs, thereby increasing mesenchymal traits and invasiveness. Our findings collectively indicate that Bcl-w functions as a positive regulator of invasiveness by inducing mesenchymal changes and that trigger their aggressiveness of glioblastoma cells.</p> </div

Bcl-w is upregulated in GBM and promotes the expression of mesenchymal-related marker proteins.

<p><b>A</b>, premade brain cancer tissue microarrays (AccuMax, A221-iv) from patients were subjected to immunohistochemical staining with anti-Bcl-w antibody (R & D systems, Minneapolis, MN). Non-neoplastic and corresponding glial tumor grade iii/iv tissues from patients (P1-P12). Bar scale, 100 µm. <b>B</b>, U251 cells were transfected with either empty pcDNA vector or that containing Bcl-w cDNA. Bcl-w expression was detected using Western blotting with β-actin as the loading control. Expression levels of mesenchymal proteins were analyzed using Western blot analysis with anti-Twist1, anti-Snail, anti-Slug, anti-vimentin, anti-E-cadherin and anti-Bcl-w in control vector and Bcl-w-overexpressing cells. <b>C</b>, U251 cells were transfected with control or siRNA oligonucleotides targeting Bcl-w (20nM) for 24 hours. Transfected control or si-Bcl-w U251 cells were subjected to Western blot analysis with the indicated antibodies. <b>D</b>, confocal microscopy analysis of vector or Bcl-w-overexpressing cells showing Bcl-w (Red, Alexa 568) and vimentin (Green, Alexa 488) and DAPI (Blue). Scale bar, 50 µm.</p

Bcl-w contributes to invasiveness in various cancer cell types including U373, U87MG, MDA-MB-231 and H1299.

<p>Cell lysates were prepared using vector- and Bcl-w-transfected cells, and Western blotting performed to compare p-GSK3β, β-catenin and MMP-2 protein levels. Transfectants were additionally subjected to Matrigel invasion assays. *, <i>p</i>< 0.01, ***, <i>p</i>< 0.0005 versus vector control, n = 5.</p

Activation of MMP-2 and FAK mediates Bcl-w-induced invasion upstream of FAK.

<p><b>A</b>, right image, MMP-2 siRNA (20nM) was introduced into the indicated U251 transfectants, and after 24 hours of incubation, p-FAK (Y397) and MMP-2 protein levels were compared using Western blotting. Left image, invasion assays were performed using small interfering RNA MMP-2-treated and untreated cells. *, p< 0.01 versus untreated control, n = 5. <b>B</b>, top image, FAK siRNA was introduced into the indicated transfectants, and after 24 hours of incubation, cellular levels of FAK, p-FAK and MMP-2 compared using Western blotting. Bottom plots, invasion assays were conducted using the indicated cells. *, p< 0.05, n = 5. <b>C</b>, top images, vector- and Bcl-w-expressing cells were transiently transfected with expression vectors for HA-tagged dominant-negative FAK mutant (FAKY397F). After 24 hours of incubation, expression of the introduced mutants in cells was verified by Western blotting. Bottom plots, invasive potentials of the indicated cells were compared. *, <i>p</i>< 0.05, n = 5.</p

Twist1, Snail and vimentin regulate the invasiveness of glioblastoma cells.

<p>Down-regulation of Twist1 and Snail leads to inhibition of glioma invasion and vimentin expression. <b>A</b>, the indicated U251 cell transfectants were incubated in serum-free medium in the presence or absence of PI3K inhibitor (LY294002 (LY); 10 µmol/L) and Akt inhibitor (Akt-I; 10 µmol/L) for 1 hour. Expression levels of Twist1, snail and vimentin proteins were compared using Western blotting. <b>B</b>, control or Bcl-w-expressing U251 cells were transfected with 20nM of Twist1, Snail or vimentin siRNA for 24 hours and were subjected to Western blotting with mesenchymal-related proteins or anti-Bcl-w antibodies. <b>C</b>, cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068030#pone-0068030-g002" target="_blank">Figure 2B</a> incubated in a Matrigel-coated transwell for 20 hours. *, <i>p</i>< 0.05, **, <i>p</i>< 0.005, n = 5.</p

Bcl-w enhances transloation of β-catenin into the nucleus in U251 glioblastoma cells.

<p><b>A</b>, levels of p-Akt, p-GSK3β, GSK3β, p-β-catenin, β-catenin and TCF-4 in cell lysates were compared by Western blotting using β-actin as a loading control. Conditioned media were prepared by incubating the vector and Bcl-w transfectants in serum-free medium for 24 hours. MMP-2 and MMP-9 activities were compared using zymography. Protein loading volumes were verified with Ponceau S staining. <b>B</b>, levels of β-catenin protein that translocated into the nucleus and Bcl-w protein in vector- or Bcl-w-transfected U251 cells were examined using confocal microscopy. Cells were stained with anti-β-catenin (green) or anti-Bcl-w (red) antibody, followed by nuclear staining with DAPI (blue). Scale bar, 50 µm. <b>C</b>, after separation of cells into cytoplasm and nuclear fractions for the indicated transfectants, each fraction was subjected to Western blotting with anti-β-catenin, anti-Lamin A/C (nucleus marker) and anti-β-actin (cytoplasm marker) antibodies.</p

Bcl-w-induced β-catenin signaling components promote invasive potential via activation of the PI3K-Akt pathway.

<p><b>A</b>, the indicated U251 cell transfectants were incubated in serum-free medium in the presence or absence of PI3K inhibitor (LY294002 (LY); 10 µmol/L) or Akt inhibitor (Akt-I; 10 µmol/L) for 1 hour. Expression levels and activities of p-Akt, p-GSK3β, β-catenin, TCF-4 and MMP-2 proteins were compared using Western blotting. <b>B</b>, cells treated with PI3K inhibitor or Akt inhibitor in the lower compartments of the invasion chambers for 24 hours, respectively. Invasive potential of treated cells was compared. *, p< 0.05 versus untreated control, n = 5. <b>C</b>, β-catenin and TCF-4 siRNAs (20nM) were introduced into vector or Bcl-w overexpressing cells, and cellular levels of β-catenin, TCF-4, MMP-2 and p-FAK compared after 24 hours of incubation using Western blotting with β-actin as a loading control. <b>D</b>, invasive potential of the indicated transfectants was compared. *, p< 0.05, n = 5.</p

PTRF/Cavin‑1 is Essential for Multidrug Resistance in Cancer Cells

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts

PTRF/Cavin‑1 is Essential for Multidrug Resistance in Cancer Cells

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts