Evangelia Patsavoudi

A Critical Role for HSP90 in Cancer Cell Invasion Involves Interaction with the Extracellular Domain of HER-2

HSP90 is a ubiquitously expressed molecular chaperone that controls the folding, assembly, intracellular disposition, and proteolytic turnover of many proteins, most of which are involved in signal transduction processes. Recently, a surface form of HSP90 has been identified and associated with cell migration events. In this paper, we explore the interaction of surface HSP90 with HER-2, a receptor-like glycoprotein and member of the ErbB family of receptor tyrosine kinases that play central roles in cellular proliferation, differentiation, and migration as well as in cancer progress. The involvement of HSP90 in the regulation of HER-2 has been attributed so far to receptor stabilization via interaction with its cytoplasmic kinase domain. Here we present evidence, using glutathione S-transferase pull-down and transfection assays, for a novel interaction between surface HSP90 and the extracellular domain of HER-2. Specific disruption of this interaction using mAb 4C5, a function-blocking monoclonal antibody against HSP90, inhibits cell invasion accompanied by altered actin dynamics in human breast cancer cells under ligand stimulation conditions with heregulin. Additionally, disruption of surface HSP90/HER-2 interaction leads to inhibition of heregulin-induced HER-2-HER-3 heterodimer formation, reduced HER-2 phosphorylation, and impaired downstream kinase signaling. Interestingly, this disruption does not affect HER-2 internalization. Our data suggest that surface HSP90 is involved in heregulin-induced HER-2 activation and signaling, leading to cytoskeletal rearrangement, essential for cell invasion.

HSP90 Inhibitors: Current Development and Potential in Cancer Therapy

In the last decade, the molecular chaperone HSP90 has emerged as an important target in cancer therapeutics and has subsequently become the focus of several drug discovery and development efforts. The first-in-class HSP90 inhibitor 17-AAG entered into Phase I clinical trial in 1999. Today 13 HSP90 inhibitors representing multiple drug classes, with different modes of action, are undergoing clinical evaluation. The present review will highlight the involvement of HSP90 in regulating and maintaining the transformed phenotype, provide an overview on current HSP90 inhibitors and further update on the most relevant patents which have recently appeared in the literature.

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

BackgroundHeat 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.ResultsOur 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 in vivo studies revealed that mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells into the lungs of SCID mice.ConclusionBoth 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.

Extracellular HSP90: conquering the cell surface.

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone, assisting intracellularly in the folding and conformational regulation of a multitude of client proteins that play a crucial role in growth, cell survival and developmental processes(1). Moreover HSP90 interacts with a great number of molecules that are involved in the development and/or survival of cancer cells, allowing mutant proteins to retain or gain function while permitting cancer cells to tolerate the imbalanced signaling that such oncoproteins create (2,3). Prime examples include the HER-2 receptor, c-Raf-1, Akt/PKB, CDK4, and mutant p53 (4,5). Highly specific inhibitors of HSP90 have been identified and are currently under clinical evaluation. These include geldanamycin and its derivatives 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxygeldanamycin, which inhibit cancer cell proliferation in vitro and tumor growth in vivo (6-9).

Monoclonal Antibody 4C5 Immunostains Human Melanomas and Inhibits Melanoma Cell Invasion and Metastasis

Purpose: Tumor cell metastasis constitutes a major problem in the treatment of cancer. Because the cure rate of metastatic tumors is very low, new therapeutic approaches are needed. Heat shock protein 90 (HSP90) is a molecular chaperone that is recognized as a new target for the treatment of cancer. Here, we examine the value of a monoclonal antibody (mAb) against HSP90, mAb 4C5, as a potential marker in malignant melanomas. Moreover, we investigate the possibility to use mAb 4C5 as an inhibitor of melanoma cell invasion and metastasis. Experimental Design: Paraffin blocks of formalin-fixed human melanoma tumor tissues were used to prepare tissue microarrays. The B16 F10 melanoma cell line was used in all the in vitro experiments. To assess melanoma cell invasion, the wound-healing assay and the Matrigel invasion assay were applied. To evaluate the effect of mAb 4C5 on tumor metastasis, we used an experimental model of metastatic melanoma. Results: Immunohistochemical studies done on a panel of malignant melanomas showed positive immunostaining with mAb 4C5 in all cases. mAb 4C5 inhibits B16 F10 cell invasion by binding to surface HSP90 because it is not internalized. mAb 4C5 significantly inhibits melanoma metastasis in C57BL/6 mice inoculated with B16 F10 cells. Conclusions: mAb 4C5 could be potentially used as a novel specific marker for malignant melanomas. mAb 4C5 inhibits melanoma cell invasion in vitro by binding to cell surface HSP90 expressed on B16 F10 melanoma cells. Finally, this antibody significantly inhibits melanoma metastasis, thus rendering it a potential therapeutic agent for the treatment of cancer metastasis.

Neuron- and myelin-specific monoclonal antibodies recognizing cell-surface antigens of the central and peripheral nervous system

Immunohistochemical screening of monoclonal antibodies raised against Triton X-114-treated synaptic membranes revealed two monoclonal antibodies, namely BM88 and BM72, with characteristic binding specificities in the central and peripheral nervous systems of the pig. Monoclonal antibody BM88 was exclusively associated with neuronal elements while BM72 was myelin-specific. Thus, in the central nervous system, immunostaining with BM88 was observed throughout the gray matter of all regions of the forebrain and spinal cord tested. In the peripheral nervous system, BM88 strongly labelled the perikarya and processes of dorsal root ganglion neurons as well as the myelinated and unmyelinated neuronal processes of the dorsal roots; BM88 immunoreactivity was also detected in neuronal cell bodies and fibres of the enteric ganglia. In addition, BM88 immunolabelled the cell-surface of cultured neurons derived from brain. In mixed cultures the staining was uniformly distributed on the perikarya and along the neurites of these cells. However, in neuron-enriched cultures where 95% of the cells were immunochemically identified as neurons, the staining of the neuronal surface membrane was patchy. This phenomenon was independent of days in culture and suggested that the distribution of the BM88 antigen on the cell surface of neurons may be regulated by neuron glia interactions. By Western blotting, the antigen recognized by BM88 in brain membrane fractions which had undergone reducing sodium dodecyl sulphate/polyacrylamide gel electrophoresis was shown to be a 22,000 mol. wt polypeptide. When extracted with Triton X-114 this polypeptide partitioned into the detergent-rich phase, a property typical of an amphipathic membrane protein. In non-reducing conditions BM88 bound to a band with a molecular weight of 43,000. These results show that the BM88 antigen is composed of two polypeptide chains of equal molecular weight linked by disulphide bridges. Monoclonal antibody BM72 recognized a myelin-associated antigen in the central and peripheral nervous system. Immunohistochemical evidence suggested a cell-surface location for this antigen. By solid phase radioimmunoassay, monoclonal antibody BM88 was shown to cross-react with brain membrane fractions from pig, rabbit and rat while BM72 recognized only a pig membrane antigen. Both monoclonal antibodies BM88 and BM72 may be used as specific cellular markers in the nervous system.

Cell surface Cdc37 participates in extracellular HSP90 mediated cancer cell invasion.

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 HSP90s 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.

Purification and Characterization of Neuron-Specific Surface Antigen Defined by Monoclonal Antibody BM88

Abstract: Monoclonal antibody BM88 recognizes a neuronspecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22‐kDa polypeptide by reducing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X‐100 a monomeric structure was implied. N‐Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase‐separation experiments with Triton X‐114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3‐kDa membrane‐associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.

Inhibiting matrix metalloproteinases, an old story with new potentials for cancer treatment.

Five decades of extensive research have passed since the description for the first time of the existence of an enzyme, which had the ability to degrade collagen during the metamorphosis of tadpoles. In fact, during these years, a large family of enzymes possessing the unique ability of degrading the extra cellular matrix (ECM) has been discovered. These enzymes are widely known as Matrix Metalloproteinases (MMPs) and it is noteworthy that many members of this family are directly linked to several human diseases such as arthritis and cancer. Moreover, due to the critical role of certain members of MMPs in cancer invasion and metastasis, great efforts have been made in order to find new inhibitory compounds against these MMPs. In this work we attempt to summarize the current status of the intervention strategies against MMPs, using inhibitory compounds that could block the activation of MMPs directly or indirectly. Furthermore we will try to shed light towards new potential strategies of MMP inhibitors using monoclonal antibodies.

Identification of a novel neuron-specific surface antigen in the developing nervous system, by monoclonal antibody 4C5

Monoclonal antibody 4C5 was obtained after immunization of Balb/c mice with a crude membrane preparation derived from the brains of 15-day-old rat embryos. As revealed by immunocytochemistry on primary cell cultures from embryonic rat brain, it was shown that the antigen recognized by monoclonal antibody 4C5 (4C5 antigen) is localized on the cell surface of the neurons. Preliminary biochemical characterization showed that it is a peripheral protein with a molecular weight of 94,000. The 4C5 antigen does not appear to be linked with other polypeptides by S--S bonds and contains few or no disulphide intramolecular bridges. N-Glycanase digestion indicated that the protein is probably not glycosylated. Monoclonal antibody 4C5 crossreacts with membrane fractions from rat, rabbit, pig and human developing brain. It was shown by immunohistochemistry that the 4C5 antigen is widely distributed in the embryonic and adult rat brain. In the peripheral nervous system 4C5 immunoreactivity was present in dorsal root ganglion neurons. Immunoblotting and immunohistochemistry on dissociated cells from rat brain and on tissue sections of brain and dorsal root ganglia revealed an age-dependent decline in the expression of the epitope recognized by monoclonal antibody 4C5, in the central and peripheral nervous system. In particular, intense 4C5 immunoreactivity was observed during the embryonic and early postnatal ages. By the second postnatal week, expression of the protein was greatly reduced, becoming very weak at later stages of development and in the adult animal. In PC12 cell cultures, expression of the 4C5 antigen was intense in proliferating cells while being greatly reduced after nerve growth factor induced differentiation of these cells. The increased expression of the 4C5 antigen in proliferating PC12 cells and the prominent presence of this molecule during a time of neuronal migration suggest that it is involved in these developmental events.