Cdc37 is localized on the cell surface of MDA-MB 453 and MDA-MB 231 breast cancer cells.

<p><b>A</b>, Indirect immunofluorescence of live MDA-MB 453 cells using anti-Cdc37 antibody. The immunolabeling reveals the surface localization of Cdc37. Anti-HER2 and mAb 4C5 were used as positive controls. Anti –EGFR antibody was used as negative control. <b>B</b>, Indirect immunofluorescence of live MDA-MB 231 cells using anti-Cdc37 antibody. Anti-EGFR and mAb 4C5 were used as positive controls. Anti –HER2 antibodies was used as negative control. <b>C</b>, Cell fractionization of MDA-MB 453 cells followed by Western blot analysis confirmed the presence of Cdc37 in cell membrane fractions. Anti-HER2 antibodies were used as positive and negative controls in the membrane and cytosolic fractions respectively. <b>D</b>, Cell fractionization of MDA-MB 231 cells followed by western blot analysis confirmed the presence of Cdc37 on cell membrane fractions. Anti-EGFR antibodies were used as positive and negative controls in the membrane and cytosolic fractions respectively. In C and D antibody against the intracellular protein Cyclin D1 gave positive and negative immunostaining in the cytosolic and membrane fractions of both cell lines, respectively. CF, cytosolic fraction; MF, membrane fraction. Scale bar = 20 µm.</p

Cdc37 is involved in MDA-MB 453 cell invasion.

<p><b>A</b>, Wound healing assay. Images obtained at 0 hours and 24 hours after scratch formation. Cells were let to migrate in control conditions or in the presence of 200 µg/ml of anti-Cdc37 antibody. <b>B</b>, At the end of the time point cells were stained with Trypan Blue. No significant cell death is observed in both cases. <b>C</b>, Quantitive effect of Cdc37 in cell migration. The addition of 200 µg/ml of anti-Cdc37 antibody in the cell culture medium resulted in a 57,7% inhibition of wound closure when compared to the control that was considered as 100% wound closure (P<0.005). Column is the average of three independent experiments ± SE. Within a single experiment each condition was tested in triplicate. Scale bars A = 165 µm, B = 80 µm.</p

Specificity of the commercial anti-Cdc37 antibody and confirmation of the cell surface localization of Cdc37, using siRNA technology targeting Cdc37.

<p>A, B, immunofluorescence of live MDA-MB 453 and MDA-MB 231 cells respectively, using anti-Cdc37 antibody. Cells were transfected with siRNA targeting Cdc37 (shRNA Cdc37) or with plasmid DNA without any insertion (control). ShRNA Cdc37 transfected cells exhibit extremely low levels of surface immunofluorescence staining as compared to controls. Scale bar = 20 µm. C, D, Western blot analysis of total cell lysates derived from MDA-MB 453 and MDA-MB 231 cells respectively, using the anti-Cdc37 antibody, in cells transfected with siRNA targeting Cdc37 and control cells. The level of Cdc37 detected is significantly lower in both cell lines transfected with siRNA targeting Cdc37, when compared with control transfections.</p

Cell Surface Cdc37 Participates in Extracellular HSP90 Mediated Cancer Cell Invasion

<div><p>Cdc37 is a 50 kDa molecular chaperone which targets intrinsically unstable protein kinases to the molecular chaperone HSP90. It is also an over-expressed oncoprotein that mediates carcinogenesis and maintenance of the malignant phenotype by stabilizing the compromised structures of mutant and/or over-expressed oncogenic kinases. Here we report that Cdc37 is not restricted intracellularly but instead it is also present on the surface of MDA-MB-453 and MDA-MB-231 human breast cancer cells, where it is shown to participate in cancer cell motility processes. Furthermore, we demonstrate using an anti-Cdc37 cell impermeable antibody, that similarly to its intracellular counterpart, this surface pool of Cdc37 specifically interacts with HSP90 as well as the kinase receptors HER2 and EGFR on the cell surface, probably acting as a co-factor in HSP90's extracellular chaperoning activities. Finally, we show that functional inhibition of surface HSP90 using mAb 4C5, a cell impermeable monoclonal antibody against this protein, leads not only to disruption of the Cdc37/HSP90 complex but also to inhibition of the Cdc37/ErbB receptors complexes. These results support an essential role for surface Cdc37 in concert with HSP90 on the cell surface during cancer cell invasion processes and strengthen the therapeutic potential of mAb 4C5 for the treatment of cancer.</p> </div

The anti-Cdc37 antibody is cell impermeable.

<p>Anti-Cdc37 antibody internalization was tested in both MDA-MB 453 and MDA-MB 231 cells. Live cells from the two cell lines were grown on coverslips and incubated at 37°C for 16 h with anti-Cdc37. MAb 4C5 and anti-HSP90 were used as positive and negative controls respectively. For detection of antibody internalization, cells were fixed and permeabilized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042722#s2" target="_blank">materials and methods</a> and antibodies were detected by confocal microscopy using an Alexa 488 secondary antibody. No internalization of the anti-Cdc37 antibody was observed even in both cell lines. Scale bar = 20 µm.</p

Cell surface interaction of Cdc37 and HSP90.

<p>A, Membrane protein fractions from MDA-MB 453 and MDA-MB 231 cells (control) were immunoprecipitaded with anti-Cdc37 antibody. Analysis of bound proteins by Western blot with anti HSP90 antibody and anti HER-2 and anti –EGFR antibodies respectively, revealed that Cdc37 interacts not only with HSP90 but also with both ErbB receptors studied. Presence of 200 µg/ml of the cell impermeable anti-Cdc37 for 16 h in the culture medium of the two cell lines followed by immunoprecipitation and Western blot analysis as above revealed that the anti-Cdc37 antibody disrupts the association of Cdc37 with HSP90 and the ErbB receptors in the two cell lines. <b>B</b>, MDA-MB 453 and MDA-MB 231 cells were treated or not with 200 µg/ml mAb 4C5 for 16 hours. Membrane protein fractions derived from these cultures were immunoprecipitaded with anti-Cdc37 followed by Western blot analysis using anti-HSP90 and anti-HER2 and anti EGFR antibodies respectively. <b>C</b>, Membrane protein fractions from MDA-MB 231 cells cultured in the absence or presence of 200 µg/ml of mAb 4C5 for 16 h were immunoprecipitated with the anti-EGFR antibody. Analysis of bound proteins by western blot with anti-HSP90 antibody revealed that surface HSP90 interacts with EGFR and that this interaction is disrupted by mAb 4C5. Irrelevant IgGs served as negative control for all the above experiments. <b>D</b>, Densitometry quantification of the observed reduced interactions in the presence of anti-Cdc37 in the culture medium of MDA-MB 453 and MDA-MB 231 cells resulted in a 33% and 59% decrease with HSP90 and HER-2 respectively regarding the MDA-MB 453 cells and in a 56% and 47% decrease with HSP90 and EGFR respectively regarding the MDA-MB 231 cells. Presence of mAb 4C5 in the culture medium of MDA-MB 453 and MDA-MB 231 cells resulted in a 60% and 58% decrease with HSP90 and HER-2 respectively regarding the MDA-MB 453 cells and in a 88% and 70% decrease with HSP90 and EGFR respectively regarding the MDA-MB 231 cells.</p

ch-4C5 inhibits the metastatic deposition of MDA-MB-453 cancer cells into the lungs of SCID mice.

<p>Control or ch-4C5 treated MDA-MB-453 cells were labeled with the fluorescent dye DiO and injected into SCID mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023906#s4" target="_blank">Materials and Methods</a>. Evaluation of metastatic deposits was performed several hours later. A. Representative cryosections of the lungs of control and ch-4C5 treated mice. The arrows show MDA-MB-453 cells stained with DiO present in the lung tissue. A significant decrease in the deposition of cancer cells was observed in the lungs of ch-4C5 treated mice. Scale bar: 100 µm <b>B</b>. Quantitative effect of ch-4C5 on the metastatic deposition of MDA-MB-453 cells into the lungs, showed a 38% inhibition when compared to control animals. The bars represent the average of two independent experiments ±SEM. Statistical significance of differences was tested by Student's t test (p<0.01).</p

ch-4C5 inhibis cancer cell invasion in <i>vitro</i>.

<p><b>A</b>. Wound healing assay. Photographs represent phase-contrast images obtained at zero time (left panel) and at 24 h (right panel) after scratch formation, showing MDA-MB-453 cell migration either in control cultures or cultures including anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5. Scale bar: 200 µm. <b>B</b>. Quantitative effect of antibodies on the closure of the wound. Addition of 200 µg/ml of anti-HSP90α and mAb 4C5 in the culture medium resulted in a 48% and 55% reduction of wound closure, respectively when compared to control cultures that were considered as resulting in 100% wound closure. Addition of 200 µg/ml rec-4C5 and ch-4C5 in the culture medium resulted in a 46% and 46% inhibition of wound closure, respectively. Statistical significance of differences was assessed by Student's t test. The presence of anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5 had a statistically significant effect on the wound closure (p<0.01 in each case). <b>C</b>. Phase-contrast images obtained at zero time (left panel) and at 24 h after scratch formation (right panel), showing B16 F10 melanoma cell invasion in a wound healing assay in the presence of 200 µg/ml of ch-4C5. Scale bar: 200 µm. <b>D</b>. Quantitative effect of increasing concentrations of ch-4C5 on the invasion level of B16 F10 melanoma cells. Presence of 50 µg/ml ch-4C5 resulted in 15% inhibition of invasion, while addition of 100 µg/ml and 200 µg/ml ch 4C5 resulted in 21% and 43% inhibition of migration, respectively when compared to control cultures that were considered as resulting in 100% wound closure. <b>E</b>. Visualization of dead cells using trypan blue dye. In ch-4C5 treated cultures, the cell death incidence is similar to that observed in the control cultures. In contrast, in the cultures treated with the anti-HSP90α antibody, a much greater number of cells are stained with Trypan blue, indicating an increased incidence of cell death Scale bar, 30 µm. <b>F</b>. Control and ch-4C5 treated cells were fixed permeabilized and stained with fluorescently labelled phalloidin. Scale bar 40 µm <b>G</b>. Higher magnification showing phalloidin staining (F-actin). Ch-4C5 effectively blocks spreading of lamellipodia. Scale bar: 16 µm.</p

ch-4C5 binds to the surface pool of HSP90 and is not internalized by MDA-MB-453 cells.

<p><b>A</b>. Immunofluoresence labelling of MDA-MB-453 cells using both the rec-4C5 and ch-4C5. The punctuate immunolabeling indicates the surface pool of HSP90. Negative controls were performed using an antibody against the intracellular protein β tubulin (data non shown). Scale bar: 20 µm <b>B</b>. Immunofluoresence detection of intracellular HSP90 in fixed MDA-MB-453 cells, permeabilized with 0.1% Triton X-100. Similarly to the murine antibody, rec-4C5 and ch-4C5 recognize the intracellular pool of HSP90. Scale bar: 20 µm <b>C</b>. For detection of antibody internalization, live cells were incubated with the antibodies for various time intervals, then fixed, permeabilized and fluorescently labelled. No internalization of mAb4C5, rec-4C5 and ch-4C5 is observed even after 24 h of incubation. In contrast the anti-HSP90α antibody could be detected intracellularly after 24 h of incubation. Scale bar: 20 µm <b>D</b>. MDA-MB-453 cell lysates, treated with mAb 4C5, rec-4C5, ch-4C5 and anti-HSP90α were analyzed by western blot using antibodies against ErbB2, Akt, cRaf and HSP90α. Actin served as a loading control. Presence of mAb 4C5, rec-4C5 and ch-4C5 did not affect the levels of the kinases compared to controls. In contrast the cell permeable anti-HSP90α antibody significantly reduced the levels of ErbB2, Akt and cRaf.</p

Electrophoretic mobility and specificity of antibodies.

<p><b>A</b>. <i>Left panel:</i> SDS-PAGE of purified antibodies under reducing conditions followed by Coomasie Brilliant Blue-R staining revealed in all cases an approximately 25 kDa band corresponding to the L-chain. <i>Right panel:</i> Under non-reducing conditions the antibodies are shown to migrate as a L-chain dimer. <b>B</b>. Western blot of MDA-MB-453 cell lysates using mAb 4C5, rec-4C5, ch-4C5 and a commercial anti-HSP90α antibody, serving as positive control. In all cases a single 90 kDa immunoreactive band corresponding to HSP90 is observed. <b>C</b>. Immunoprecipitation in MDA-MB-453 cell lysates with anti-HSP90α, followed by immunoblotting with either the murine or the recombinant antibodies. In all cases a single immunoreactive band is observed. <b>D</b>. Reverse immunoprecipitation experiments in MDA-MB-453 cell lysates using mAb 4C5, rec-4C5 and ch-4C5, followed by western blot with anti-HSP90α. In all cases a single immunoreactive band is observed indicating that both recombinant antibodies recognize HSP90.</p