Monoclonal antibody 4C5 prevents activation of MMP2 and MMP9 by disrupting their interaction with extracellular HSP90 and inhibits formation of metastatic breast cancer cell deposits

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 90 (HSP90) is a molecular chaperone that is considered a new target for the treatment of cancer. Increasing data reveal an extracellular chaperoning activity for HSP90. Here we investigate the interaction of the secreted isoforms of HSP90 with matrix metalloproteinases (MMP) MMP2 and MMP9. Moreover we examine the role of a monoclonal antibody (mAb) against HSP90, mAb 4C5, regarding these interactions and its value as a potential inhibitor of human breast cancer cell invasion and metastasis.</p> <p>Results</p> <p>Our results showed that both HSP90α and HSP90β are secreted by MDAMB453 human breast cancer cells and interact with MMP2 and MMP9. MAb 4C5, while not affecting the secretion of inactive MMPs, inhibits their activation by disrupting their interaction extracellularly with both isoforms of HSP90. The <it>in vivo </it>studies revealed that mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells into the lungs of SCID mice.</p> <p>Conclusion</p> <p>Both isoforms of HSP90 are secreted by MDAMB453 cells and interact with MMP2 and MMP9. MAb 4C5 prevents MMP2 and MMP9 activation, by disrupting their interaction with HSP90. Finally mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells, by preventing their extravasation and infiltration in the lung tissue and therefore it could be used as a potential therapeutic agent for cancer metastasis.</p

The 4C5 Cell-Impermeable Anti-HSP90 Antibody with Anti-Cancer Activity, Is Composed of a Single Light Chain Dimer

MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics

Extracellular Molecules Involved in Cancer Cell Invasion

Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion

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

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

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

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