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

Abstract

Background: 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.

Results: Our results showed that both HSP90alpha and HSP90beta 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 in vivo studies revealed that mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells into the lungs of SCID mice.

Conclusion: 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.

Figure 1

Both the α and the β isoforms of HSP90 are secreted by MDAMB453 cells and interact with MMP2 and MMP9 metalloproteinases. (A) Cell lysates were immunoblotted with mAb 4C5 recognizing both isoforms of HSP90 and serving as positive control. Concentrated supernatants derived from the cell cultures as described in Methods, were analyzed by Western blot using anti HSP90 α and anti HSP90 β antibodies and mAb 4C5. Anti beta actin antibody was used in both fractions. Secretion of the two isoforms of HSP90 was detected in the supernatant derived from MDAMB453 cells. The absence of beta actin in the supernatant demonstrates that there is no contamination of this fraction with the intracellular components of cells during the experimental procedure. (B) Western blot analysis of concentrated supernatants derived from MDAMB453 cell cultures, using anti-MMP2 and anti-MMP9 antibodies showed that both the pro-MMP2 and pro-MMP9 as well as their activated forms are present in the culture medium. Proteins derived from the culture supernatant were immunoprecipitated with anti-MMP2 and anti-MMP9 and bound proteins were analyzed by Western blot with anti-HSP90α and anti-HSP90β antibodies. Both isoforms of HSP90 interact with the two metalloproteinases. Irrelevant IgGs and anti-MMP2 or anti-MMP9 antibodies were used as negative and positive controls respectively. (C) Proteins derived from the MDAMB453 cell culture supernatant were immunoprecipitated with anti-HSP90α and anti-HSP90β antibodies and immunoprecipitants were analyzed by Western blot using the anti-MMP2 and anti-MMP9 antibodies. Irrelevant IgGs and anti-HSP90 α or anti-HSP90 β antibodies were used as negative and positive controls respectively. The reverse immunoprecipitation experiment showed that both isoforms of HSP90 interact with pro-MMP2 and pro-MMP9 and to a lesser extent with the activated forms of the two metalloproteinases. IP immunoprecipitation, WB Western blot.

Figure 2

MAb4C5 prevents MMP2 and MMP9 activation and disrupts their interaction with both the α and the β isoform of extracellular HSP90. (A) Metalloproteinases derived from the concentrated supernatants of control and mAb 4C5 treated cultures of MDAMB453 cells were isolated and analyzed by zymography, as described in Methods. Proteolysis was detected as a white zone in a dark field. Metalloproteinases previously isolated as described in Methods were used as markers. Activated MMP2 and MMP9 are absent in the mAb 4C5 treated cultures. (B) Proteins derived from the concentrated supernatants of control and mAb 4C5 treated cultures of MDAMB453 cells, prepared as described in Methods, were analyzed by Western blot using anti-MMP2 and anti-MMP9 antibodies. Presence of mAb 4C5 in the culture medium inhibits activation of both metalloproteinases. (C) Proteins from the above described supernatants were immunoprecipitated with anti-MMP2 and anti-MMP9 antibodies and bound proteins were analyzed by Western blot with anti-HSP90α and anti-HSP90β antibodies. MAb 4C5 disrupts the association of both isoforms of HSP90 with the two metalloproteinases, since anti-MMP2 and anti-MMP9 antibodies did not co-immunoprecipitate any detectable levels of HSP90 in the mAb 4C5 treated cultures, when compared with controls. Western blot analysis of the MMP2 and MMP9 immunoprecipitants, using the corresponding antibodies was performed as positive controls and irrelevant IgGs were used as negative controls. IP immunoprecipitation, WB Western blot.

Figure 3

MAb 4C5 inhibits the metastatic deposition of MDAMB453 cancer cells into the lungs of SCID mice. MDAMB453 cells were labeled with the fluorescent dye DiI and injected into SCID mice, in the presence either of 100 μg/ml of mAb 4C5 or of an irrelevant antibody (control), as described in Methods. Evaluation of metastatic deposits was performed several hours later. (A) Macroscopic level: An important number of metastatic deposits (arrow) was observed in control animals as compared to mAb 4C5 treated mice. (B) Microscopic level: Representative cryosections of the lungs of control and mAb 4C5 treated mice. The arrows show MDAMB453 cells stained with DiI present in the lung tissue. A significant decrease in the deposition of cancer cells was observed in the mAb4C5 treated mice. (C) Quantitative effect of mAb 4C5 on the metastatic deposition of MDAMB453 cells into the lungs showed an 86.67% inhibition of the metastatic deposition in the mAb 4C5 treated mice when compared to the control animals. (D) A 3D reconstitution of a cryosection from a control animal: The arrow demonstrates infiltration of metastatic deposits in the lung tissue. (E) MDAMB453 cells restrained in the inner surface of large pulmonary vessels derived from mAb 4C5 treated mice. On the contrary, in the lungs from control mice MDAMB453 cells could rarely be detected stagnating in the inner surface of the pulmonary vessels. However, cancer cells were clearly observed dispersed in the lung tissue. Arrows indicate the pulmonary vessels. (F) Quantitative effect of mAb 4C5 on the retention of MDAMB453 cells in the pulmonary vessels. A 15.4% of the vessels visualized in the control animals showed intravascular cancer cell retention. In contrast in the mAb 4C5 treated mice, cancer cells were observed stagnating in the inner surface of 58.76% of the calculated vessels (p < 0.01 Bar, 40 μm).