prinomastat and Breast-Neoplasms

We illustrate the use of quantitative proteomics, namely isotope-coded affinity tag labelling and tandem mass spectrometry, to assess the targets and effects of the blockade of matrix metalloproteinases by an inhibitor drug in a breast cancer cell culture system. Treatment of MT1-MMP-transfected MDA-MB-231 cells with AG3340 (Prinomastat) directly affected the processing a multitude of matrix metalloproteinase substrates, and indirectly altered the expression of an array of other proteins with diverse functions. Therefore, broad spectrum blockade of MMPs has wide-ranging biological consequences. In this human breast cancer cell line, secreted substrates accumulated uncleaved in the conditioned medium and plasma membrane protein substrates were retained on the cell surface, due to reduced processing and shedding of these proteins (cell surface receptors, growth factors and bioactive molecules) to the medium in the presence of the matrix metalloproteinase inhibitor. Hence, proteomic investigation of drug-perturbed cellular proteomes can identify new protease substrates and at the same time provides valuable information for target validation, drug efficacy and potential side effects prior to commitment to clinical trials.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Design; Drug Evaluation, Preclinical; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Isotope Labeling; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Organic Chemicals; Peptide Hydrolases; Protease Inhibitors; Protein Processing, Post-Translational; Protein Transport; Proteomics; Substrate Specificity; Systems Biology; Tandem Mass Spectrometry

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the breakdown of proteins of connective tissue. Through this action they play an important role in growth, development and tissue repair. Recent studies also suggest that MMPs are utilised in cancer, facilitating both local tumour invasion and metastasis. Levels of certain MMPs such as stromelysin-3 and gelatinase are elevated in tumour-associated stroma compared to non-involved tissue. A series of synthetic low molecular weight MMP inhibitors have been produced. Early inhibitors were based on the peptide structure of collagen, although more recently non-peptide inhibitors have also been developed. The inhibitors are selective for the MMP family and are active at low nanomolar concentrations. Experiments in models of breast cancer have shown that MMP inhibitors can significantly reduce the growth rate of both primary and secondary tumours, and can block the process of metastasis. Several MMP inhibitors have now started clinical trials in patients with advanced malignancy. Although not the optimum setting for a tumouristatic agent, early results suggest this approach may be effective in slowing tumour growth. Trials in the adjuvant setting will provide the most important test of these inhibitors and should determine their potential to complement existing cytoreductive treatments and prolong survival.

Topics: Antineoplastic Agents; Breast Neoplasms; Clinical Trials as Topic; Drug Design; Female; Humans; Metalloendopeptidases; Organic Chemicals; Protease Inhibitors

Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90alpha, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.

Topics: Amino Acid Sequence; Axl Receptor Tyrosine Kinase; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Culture Media, Conditioned; Drug Screening Assays, Antitumor; Galectin 1; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Isotope Labeling; Matrix Metalloproteinase 14; Matrix Metalloproteinase Inhibitors; Models, Biological; Molecular Sequence Data; Oncogene Proteins; Organic Chemicals; Progranulins; Protease Inhibitors; Proteomics; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Reproducibility of Results; Substrate Specificity

Macromolecular contrast medium-enhanced magnetic resonance imaging was applied to monitor the effect of matrix metalloprotease (MMP) inhibition on microvascular characteristics of human breast cancers implanted in athymic rats. Twice-daily intraperitoneal administration of Prinomastat over 1.5 days induced significant declines in MRI-assayed microvascular permeabilities (p<0.05); but this leak suppression effect had extinguished by the 10(th) day of MMP treatment using the same dose and time schedule. Results demonstrate that Prinomastat produces a rapid but transient decrease in tumor vascular permeability. Contrast-enhanced MRI using macromolecular contrast medium may prove useful as a biomarker for the dynamic MMP biological effect in cancers.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Capillary Permeability; Cell Division; Cell Line, Tumor; Contrast Media; Enzyme Inhibitors; Female; Homozygote; Humans; Magnetic Resonance Imaging; Matrix Metalloproteinase Inhibitors; Microcirculation; Neoplasm Transplantation; Neoplasms; Organic Chemicals; Rats; Rats, Nude; Statistics as Topic; Time Factors

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alphavbeta3 integrin have been directly implicated in tumor cell dissemination and metastasis. We have demonstrated that in the case of breast carcinoma MCF7 cells co-expressing MT1-MMP and alphavbeta3 integrin, the proteinase processes the pro-alphav integrin subunit, thus facilitating alphavbeta3 integrin maturation and cell migration on vitronectin. Our findings show that cell surface MT1-MMP is a short-lived protein with a life span in the range of several hours. In contrast, turnover of alphavbeta3 integrin is much slower. The half-life of alphavbeta3 heterodimer is about 24 hr. This large difference in life span allowed us to distinguish between the effects of MT1-MMP on cell migration brought by matrix proteolysis from those imposed through alphavbeta3 integrin maturation. We then modulated the enzyme's activity by a potent hydroxamate MMP inhibitor, Prinomastat (AG3340), to analyze the divergent effects of MT1-MMP on cell migration. Although Prinomastat immediately blocked MT1-MMP-mediated matrix degradation, the pool of MT1-MMP-modified alphavbeta3 integrin molecules was still capable of mediating cell-matrix interactions. To our considerable surprise, inhibition of MT1-MMP-dependent vitronectin proteolysis by Prinomastat allowed a several-fold increase in migration of MCF7 cells co-expressing MT1-MMP and alphavbeta3 integrin. In contrast, long-term Prinomastat inhibition of MT1-MMP-dependent pro-alphav cleavage and thus alphavbeta3 integrin maturation strongly inhibited cell motility. Our studies suggest that MT1-MMP could actually promote cell migration via modification of the cell surface receptors, including alphavbeta3 integrin, rather than facilitate cell migration through direct cleavage of the matrix proteins.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Movement; Enzyme Inhibitors; Humans; Integrin alphaVbeta3; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Organic Chemicals; Tumor Cells, Cultured; Vitronectin

We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP-2 and -9), MT-MMP-1 (MMP-14), and collagenase-3 (MMP-13) in many tumor models. AG3340 produced dose-dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose-response relationship was explored in three models: COLO-320DM colon, MV522 lung, and MDA-MB-435 breast. Dose-dependent inhibitions of tumor growth (over 12.5-200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen-independent PC-3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD-31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16-F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front-line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Division; Cell Line; Colonic Neoplasms; Female; Glioma; Humans; Kinetics; Lung Neoplasms; Male; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, SCID; Neovascularization, Pathologic; Organic Chemicals; Protease Inhibitors; Transplantation, Heterologous