4-aminophenylmercuriacetate and Breast-Neoplasms

Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.

Topics: Antibodies; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Collagen; Dose-Response Relationship, Radiation; Drug Combinations; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Laminin; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Neoplasm Invasiveness; Phenylmercuric Acetate; Proteoglycans; RNA, Messenger; Tissue Inhibitor of Metalloproteinases

HER2 is a ligand-less tyrosine kinase receptor of the ErbB family that is frequently overexpressed in breast cancer. It undergoes proteolytic cleavage that results in the release of the extracellular domain and the production of a truncated membrane-bound fragment, p95. We show that HER2 shedding is activated by 4-aminophenylmercuric acetate (APMA), a well-known matrix metalloprotease activator, in HER2-overexpressing breast cancer cells. The HER2 p95 fragment, which appears after APMA-induced cleavage, is phosphorylated. We analyzed 24 human breast cancer specimens, and a phosphorylated M(r) 95,000 HER2 band could be detected in some of them, which indicated that the truncated receptor is also present in vivo. The activation of HER2 shedding by APMA in cells was blocked with batimastat, a broad-spectrum metalloprotease inhibitor. Trastuzumab (Herceptin; Genentech, San Francisco, CA), a humanized monoclonal antibody directed at the HER2 ectodomain, which has been shown to be active in patients with HER2-overexpressing breast cancer, inhibited basal and induced HER2 cleavage and, as a consequence, the generation of phosphorylated p95. This inhibitory effect of trastuzumab was not shared by 2C4, an antibody against a different epitope of the HER2 ectodomain. The inhibition of basal and APMA-induced cleavage of HER2 by trastuzumab preceded antibody-induced receptor down-modulation, which indicated that the effect of trastuzumab on cleavage was not attributable to a decrease in cell-surface HER2 induced by trastuzumab. We propose that the inhibition of HER2 cleavage and prevention of the production of an active truncated HER2 fragment represent a novel mechanism of action of trastuzumab.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Humans; Metalloendopeptidases; Peptide Fragments; Phenylalanine; Phenylmercuric Acetate; Phosphorylation; Protease Inhibitors; Protein Structure, Tertiary; Receptor, ErbB-2; Thiophenes; Trastuzumab; Tumor Cells, Cultured

A novel matrix-degrading enzyme was identified from human breast cancer cells. This enzyme appears as major gelatinase in hormone-dependent breast cancer cell lines and has as an apparent molecular mass of 80 kDa on gelatin zymography. The 80-kDa enzyme has a unique metal ion specificity. In addition to calcium ions, the gelatinolytic activity can be supported by manganese and/or magnesium. Unlike 92- and 72-kDa gelatinases and other known members of the metalloproteinase family, the 80-kDa protease is not activated by p-aminophenylmercuric acetate and its gelatinolytic activity is not inhibited by tissue inhibitor of metalloproteinase 2. It is active over the pH range 7.5-9.5 with an optimum at pH 8.5. The enzyme degrades gelatin and type IV collagen. The proteolytic activity of the enzyme is inhibited by EDTA and leupeptin. These unique features clearly distinguish the 80-kDa protease from the known 92-and 72-kDa gelatinases. The expression of 80-kDa enzyme can be detected in hormone-dependent human breast cancer cell lines in vitro and in tumors grown from these cells in athymic nude mice.

Topics: Amino Acid Sequence; Breast Neoplasms; Edetic Acid; Endopeptidases; Enzyme Activation; Female; Gelatin; Gelatinases; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Pepsin A; Phenylmercuric Acetate; Substrate Specificity; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

Although the M(r) 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of M(r) 59,000 and/or M(r) 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor beta or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.

Topics: Breast Neoplasms; Carcinosarcoma; Collagen; Concanavalin A; Enzyme Induction; Fibroblasts; Humans; Matrix Metalloproteinase 9; Microbial Collagenase; Molecular Weight; Phenylmercuric Acetate; Skin; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured