batimastat and Breast-Neoplasms

Matrix metalloproteinases (MMPs) are a homologous family of enzymes that are involved in tissue remodeling and morphogenesis. Collectively, these enzymes are capable of degrading all components of the extracellular matrix, and they play an important role in normal physiologic conditions, such as wound healing and other processes involving tissue remodeling. However, increased activity of these enzymes now has been observed in a number of different pathological conditions, and it has been hypothesized that such increased activity of MMPs might play a role in the pathogenesis of these conditions. Cancer is one such condition; extracellular matrices constitute the principal barrier to tumor growth and spread, and there is growing experimental evidence that malignant tumors utilize MMPs to overcome these barriers. Consequently, inhibitors of MMPs represent an attractive target for a new class of anticancer agents. Marimastat and batimastat are potent broad-spectrum inhibitors of all major MMPs and have been shown to prevent or reduce spread and growth of a number of different malignant tumors in numerous animal models. Both agents are now in advanced clinical testing in a number of different solid tumors in North America and Europe. The purpose of this paper is to review available preclinical and emerging clinical data, using batimastat and marimastat as prototype MMP inhibitors in the cancer area.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Colorectal Neoplasms; Enzyme Inhibitors; Female; Hemangioma; Humans; Hydroxamic Acids; Melanoma; Metalloendopeptidases; Mice; Ovarian Neoplasms; Pancreatic Neoplasms; Phenylalanine; Protease Inhibitors; Skin Neoplasms; Thiophenes

Macrophage recruitment is essential for tissue homeostasis but detrimental in most cancers. Tumor-associated macrophages (TAMs) play a key role in cancer progression. Controlling their migration is, thus, potentially therapeutic. It is assumed that macrophages use amoeboid motility

Topics: Amides; Animals; Breast Neoplasms; Cell Movement; Female; Humans; Immunotherapy; Macrophages; Matrix Metalloproteinases; Mesoderm; Mice, Inbred C57BL; Mice, Transgenic; Microscopy; Organ Culture Techniques; Otitis; Phenylalanine; Protease Inhibitors; Pyridines; rho-Associated Kinases; Thiophenes

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1‑dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

Topics: Anti-Arrhythmia Agents; Breast Neoplasms; Cathepsin B; Cation Transport Proteins; Cell Line, Tumor; Cell Surface Extensions; Extracellular Matrix; Female; Guanidines; Humans; Hydrogen-Ion Concentration; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neoplasm Invasiveness; Peptide Hydrolases; Phenylalanine; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfones; Thiophenes

Breast cancer cells can switch from estrogen receptor α (ER)- to human epidermal growth factor receptor (HER)-driven cell growth upon acquiring antiestrogen resistance. HER ligands are cleaved by metalloproteinases leading to release of active HER ligands, activation of HER receptors and consequently increased cell growth. In this study, we investigated the importance of HER receptors, in particular HER3, and HER ligand shedding for growth and signaling in human MCF-7 breast cancer cells and MCF-7-derived sublines resistant to the antiestrogen fulvestrant. The HER3/HER4 ligand heregulin 1β induced phosphorylation of HER3, Akt and Erk, and partly rescued fulvestrant-inhibited growth of MCF-7 cells. HER3 ligands were found to be produced and shed from the fulvestrant-resistant cells as conditioned medium from fulvestrant-resistant MCF-7 cells induced phosphorylation of HER3 and Akt in MCF-7 cells. This was prevented by treatment of resistant cells with the metalloproteinase inhibitor TAPI-2. Only the broad-spectrum metalloproteinase inhibitor BB-94, and not the more selective inhibitors GM6001 or TAPI-2, which inhibited shedding of the HER ligands produced by the fulvestrant-resistant cells, was able to inhibit growth and activation of HER3 and Erk in resistant cells. Compared to MCF-7, fulvestrant-resistant cells have increased HER3 phosphorylation, but knockdown of HER3 had no inhibitory effect on resistant cell growth. The EGFR inhibitor gefitinib exhibited only a minor growth inhibition, whereas the pan-HER inhibitor CI-1033 exerted growth arrest. Thus, neither HER3 nor EGFR alone are the main driver of fulvestrant-resistant cell growth and treatment should target both receptors. Ligand shedding is not a treatment target, as receptor activation occurred, independent of release of ligands. Only the broad-spectrum metalloproteinase inhibitor BB-94 could abrogate HER3 and Erk activation in the resistant cells, which stresses the complexity of the resistance mechanisms and the requirement of targeting signaling from HER receptors by multiple strategies.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; ErbB Receptors; Estradiol; Extracellular Signal-Regulated MAP Kinases; Female; Fulvestrant; Humans; MCF-7 Cells; Phenylalanine; Phosphorylation; Protease Inhibitors; Receptor, ErbB-3; Thiophenes

The presence of mesothelin (encoded by the mesothelin [MSLN] gene) in breast cancer is associated with tumour infiltration of the lymph node. This study evaluated whether MSLN overexpression promotes breast cancer cell invasiveness and metastasis.. This study evaluated the effects of overexpression of MSLN on extracellular signal-regulated kinase (ERK1/2) and matrix metalloproteinase (MMP)-9 levels, and the invasiveness and angiogenesis of the breast cancer cell line MCF-7 in vitro, and on MCF-7-derived tumour development in vivo.. MSLN overexpression significantly increased ERK1/2 and MMP9 protein levels and activity, and the invasive and angiogenic capability of MCF-7 cells, in vitro. Inhibition of ERK1/2 suppressed MMP-9 and the invasive and angiogenic capability of MSLN overexpressing MCF-7 cells. MSLN overexpression also increased MCF-7-derived tumour metastasis in vivo.. MSLN overexpression promoted the invasive potential of MCF-7 cells through ERK1/2-dependent upregulation of MMP-9; this association may have contributed to metastasis of MCF-7 cells in vivo. Mesothelin may be a useful biomarker for cancer progression and a novel therapeutic or chemopreventive target in human breast cancer.

Topics: Animals; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Lymphatic Metastasis; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; MCF-7 Cells; Mesothelin; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Neovascularization, Pathologic; Nitriles; Phenylalanine; Protease Inhibitors; Thiophenes; Transplantation, Heterologous

Association of matrix metalloprotease 9 (MMP9) to the cell membrane is considered important in tumor growth and angiogenesis. To dissect this regulatory mechanism, we generated raft and non-raft MMP9 chimeras to force membrane expression in the MCF-7 human breast carcinoma cell line. MMP9 targeting to non-raft cell surface domains rendered a constitutive active membrane MMP9 form, suggesting a contribution by the lipid environment in MMP activation. We generated human breast cancer xenograft models using MCF-7 cells overexpressing secreted and membrane-anchored MMP9. The non-raft MMP9 chimera was constitutively active at the cell membrane in xenografts, but this activation did not correlate with an increase in MMP9-induced angiogenesis. Capillary number and vessel perimeter were specifically increased only in tumors overexpressing wild-type MMP9 (the secreted form); this increase was inhibited when tumors were induced in doxycycline-treated mice. Xenografts from tumor cells overexpressing wild-type MMP9 showed increased vascular endothelial growth factor (VEGF)/VEGFR2 receptor association, which was also dependent on MMP9 activity. These observations indicate that membrane location can influence MMP9 activity in vitro and in vivo, and confirm the relevance of stromal-associated, but not tumor-bound MMP9 in mediating tumor-induced angiogenesis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Dipeptides; Doxycycline; Enzyme Activation; Humans; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Membrane Microdomains; Membrane Proteins; Neovascularization, Pathologic; Phenylalanine; Protein Binding; Protein Transport; Receptors, LDL; Recombinant Fusion Proteins; Thiophenes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

The implication of matrix metalloproteinases (MMPs) in the major stages of cancer progression has fueled interest in the design of synthetic MMP inhibitors (MMPIs) as a novel anticancer therapy. Thus far, drugs used in clinical trials are broad-spectrum MMPIs the therapeutic index of which proved disappointingly low. The development of selective MMPIs for tumor progression-associated MMPs is, thus, likely to offer improved therapeutic possibilities.. The anti-invasive capacity of a series of pyrimidine-trione derivatives was tested in vitro in a chemoinvasion assay, and the most potent compound was further evaluated in vivo in different human tumor xenograft models. The activity of this novel selective MMPI was compared with BB-94, a broad-spectrum inhibitor.. Ro-28-2653, an inhibitor with high selectivity for MMP-2, MMP-9, and membrane type 1 (MT1)-MMP, showed the highest anti-invasive activity in vitro. In vivo, Ro-28-2653 reduced the growth of tumors induced by the inoculation of different cell lines producing MMPs and inhibited the tumor-promoting effect of fibroblasts on breast adenocarcinoma cells. Furthermore, Ro-28-2653 reduced tumor vascularization and blocked angiogenesis in a rat aortic ring assay. In contrast, BB-94 up-regulated MMP-9 expression in tumor cells and promoted angiogenesis in the aortic ring assay.. Ro-28-2653, a selective and orally bioavailable MMPI with inhibitory activity against MMPs expressed by tumor and/or stromal cells, is a potent antitumor and antiangiogenic agent. In contrast to broad-spectrum inhibitors, the administration of Ro-28-2653 was not associated with the occurrence of adverse side effects that might hamper the therapeutic potential of these drugs.

Topics: Adenocarcinoma; Administration, Oral; Animals; Antineoplastic Agents; Aorta; Breast Neoplasms; Cell Line, Tumor; Disease Progression; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Humans; Inhibitory Concentration 50; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Microscopy, Fluorescence; Models, Chemical; Neoplasm Invasiveness; Neoplasm Transplantation; Neovascularization, Pathologic; Phenylalanine; Piperazines; Protease Inhibitors; Pyrimidines; Rats; Thiophenes; Time Factors; Up-Regulation

Stromelysin-3 (ST3) has the characteristic structure of matrix metalloproteinases (MMP), but its substrate specificity and pattern of expression differ markedly from that of other MMP family members. ST3 was originally isolated on the basis of its expression in primary breast cancers and has been shown to be overexpressed in virtually all primary carcinomas, suggesting that ST3 participates in the initial development of epithelial malignancies. Recent data using murine models reported that ST3 expression was able to increase tumor take by suppressing cell apoptosis. Our present goal was to set up an in vitro model in which we could study this new function. For this purpose, we analyzed survival of MCF-7 transfectants expressing either wild-type or catalytically inactive ST3 (ST3wt or ST3cat-) in three-dimensional (3-D) culture conditions by inclusion in Matrigel. In such conditions, that mimic the in vivo microenvironment, we found a marked decrease in the percentage of cell death when active ST3 was expressed (ST3wt transfectants vs. ST3cat- or vector only transfectants) as assessed by FACS and TUNEL analysis. The addition of batimastat, a broad spectrum MMP inhibitor, reversed the increased cell survival in ST3wt transfectants, confirming that ST3 enzymatic activity was required for this effect. Finally, we analyzed the expression of anti- and pro-apoptotic proteins as well as activation of cell survival pathways and we found that ST3-mediated cell survival was accompanied by activation of both p42/p44 MAPK and AKT. Our data confirm and extend the anti-apoptotic function of ST3 and provide a useful model to dissect this new role and identify new physiological substrates.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Caspase Inhibitors; Cell Survival; Collagen; Drug Combinations; Enzyme Activation; Enzyme Inhibitors; Female; Humans; In Situ Nick-End Labeling; Laminin; Matrix Metalloproteinase 11; Metalloendopeptidases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Mutation; Phenylalanine; Phosphorylation; Protein Serine-Threonine Kinases; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Thiophenes; Transfection; Tumor Cells, Cultured; Tyrosine

Breast cancer frequently leads to incurable bone metastasis. Essential requirements for the development of bone metastasis are cell-cell and cell-matrix interactions, release of bioactive growth factors and cytokines, and removal of large amounts of bone matrix. Matrix metalloproteinases (MMPs) play an important role in all of these processes, but the possibility of using synthetic MMP inhibitors to decrease bone metastasis has received little attention.. In the present study, we tested two general MMP inhibitors, BB-94 and GM6001, in a mouse model of breast cancer-induced bone metastasis.. In a simulation of intervention therapy, mice were inoculated with breast cancer cells, and at the time of diagnosis of osteolytic lesions, the mice were treated for 10 or 15 consecutive days with BB-94 or GM6001, respectively. Both inhibitors reduced the growth of osteolytic lesions by >55% compared with control mice. Next, we simulated prevention therapy by initiating treatment with GM6001 at time of inoculation with cancer cells or 3 days earlier. Assessment of osteolytic lesions 28 days after inoculation showed that, in both cases, the treatment reduced the size of the osteolytic lesions by 60%, compared with that of control mice. Importantly, MMP inhibition also resulted in extension of symptom-free survival in the mice, whether the treatment was initiated at the time of diagnosis of osteolytic lesions or of cancer cell inoculation.. The present study suggests the potential of synthetic MMP inhibitors as intervention or prevention treatments of breast cancer-induced osteolysis.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Cell Survival; Dipeptides; Female; Genetic Therapy; Humans; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Osteolysis; Phenylalanine; Protease Inhibitors; Thiophenes; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Bone resorption is a dominant feature of many bone metastases and releases factors from the bone matrix that can promote the expression of the metastatic phenotype in cancer cells. Since proteolytic enzymes, including matrix metalloproteinases (MMPs) contribute to bone destruction by metastatic tumour cells and host cells, we have examined the effect of a MMP inhibitor, batimastat, on the ability of MDA-MB-231 cells to degrade bone in vitro and to form bone metastases in BalbC nu/nu mice. In vitro, the neoplastic cells produced MMP-2 and MMP-9, degraded [3H]-proline-labelled osteoblast matrices, and formed resorption pits in cortical bone. These phenomena were inhibited by < or = 20 microM batimastat. To induce vertebral and long bone metastases in vivo, 1x10(5) MDA-MB-231 cells were injected into the arterial circulation of BalbC nu/nu mice. Test groups were also given 30 mg/kg batimastat intraperitoneally (i.p.). After 21 days, the long bone metastases were characterised by a 67% reduction of metaphyseal medullary bone and complete replacement of marrow by tumour. In tumour-bearing mice that had been treated with 30 mg/kg batimastat i.p., the tumour volume decreased 8-fold, osteolysis was inhibited by 35%, and replacement of the bone marrow by tumour was inhibited by 65%. Similar effects were observed in the vertebral metastases. These data provide evidence that MDA-MB-231 cells can degrade osteoblast matrices and mineralised bone in vitro and support the hypothesis that MMPs are involved in the pathogenesis of osteolytic bone metastases in vivo. They demonstrate that an agent which inhibits proteolysis can retard the development of osteolytic bone metastases in this model.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Humans; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred BALB C; Phenylalanine; Thiophenes; Tumor Cells, Cultured

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

The matrix metalloprotease (MMP) family of enzymes and the urokinase plasminogen activator (uPA) pathway have both been implicated in tumor invasion and metastasis and in poor prognosis of cancer. We have previously shown that treatment with batimastat, a synthetic MMP inhibitor, leads to significant retardation but not regression of tumor growth in a human breast cancer xenograft model. In addition, batimastat treatment did not inhibit local tumor invasion, nor did it encourage stromal encapsulation of the tumor, suggesting the additional involvement of non-MMP proteolytic mechanisms. To investigate the presence of an alternative extracellular matrix protease whose activity is known to be important in breast cancer, but which is not inhibited by batimastat, expression of murine and human uPA were examined by in situ hybridization and ELISA. No differences were observed between untreated and batimastat-treated tumors regarding human uPA mRNA and protein. In contrast, murine uPA mRNA expression was increased at the tumor-stromal junction in batimastat-treated tumors in comparison with the control tumors. In agreement with these results, batimastat treatment was shown to significantly induce murine uPA protein content in the tumors. Inoculating MDA435/LCC-6 cells into immunodeficient, uPA-deficient mice resulted in tumor growth retardation as compared to tumor growth in littermate wild-type controls, while addition of batimastat treatment to uPA-/- mice did not result in further growth inhibition. The increased expression of stromal uPA may represent a cellular response to MMP inhibition and may demonstrate a new level of plasticity in the malignant progression of the disease. These results may have important implications for the clinical applications of MMP inhibitors, as well as for development of other anti-invasion drugs.

Topics: Animals; Blotting, Northern; Breast Neoplasms; DNA Primers; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Metalloendopeptidases; Mice; Mice, Nude; Phenylalanine; Plasminogen Activators; Polymerase Chain Reaction; Protease Inhibitors; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Stromal Cells; Thiophenes; Xenograft Model Antitumor Assays

The alpha 3 beta 1 integrin is elevated in several types of metastatic tumor and has been associated with increased migration and invasion. Our analysis of a series of mammary carcinomas of different histotypes and their corresponding metastases demonstrated significantly increased expression of alpha 3 beta 1 in the tumor metastases. We therefore studied alpha 3 beta 1 expression of several human breast carcinoma cell lines and its association with the invasive phenotype. The MDA-MB-231 cell line expressed high levels of the beta1, alpha 2, alpha 3, alpha 5, and alpha 6 integrin subunits along with moderate levels of the alpha v beta 3 integrin. This line was highly migratory and the most invasive using a chemo-invasion assay. In contrast, the other lines tested, MDA-MB-145, MCF-7, and SK-BR-3, showed lower migratory and invasive activity and reduced alpha 3 integrin subunit expression. Metalloproteases capable of degrading collagen IV are necessary for the invasive process. RT-PCR showed that MDA-MB-231 cells expressed MMP-9, but not MMP-2, gelatinase/collagenase IV. Gelatin zymography demonstrated that invading MDA-MB-231 cells released high levels of MMP-9 gelatinase activity. A direct role for this gelatinase in MDA-MB-231 cell invasion was confirmed by inhibition of invasion using the metalloprotease inhibitor Batimastat. Treatment of MDA-MB-231 cells with a function-blocking anti-alpha 3 antibody strongly inhibited migration and invasion. This correlated with a marked reduction in MMP-9 activity produced by MDA-MB-231 cells, suggesting a role for alpha 3 beta 1 ligand binding in cell signaling and regulation of extracellular matrix degradation.

Topics: Adult; Aged; Antibodies, Monoclonal; Breast Neoplasms; Chemotaxis; Collagen; Enzyme Induction; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha3beta1; Integrins; Ligands; Matrix Metalloproteinase 9; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Phenotype; Phenylalanine; Protease Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Thiophenes; Tumor Cells, Cultured

HER2/neu, a Mr 185,000 tyrosine kinase receptor that is overexpressed in breast cancer, undergoes proteolytic cleavage of its extracellular domain (ECD). In contrast with other membrane-bound proteins, including growth factor receptors, that are cleaved by a common machinery system, we show that HER2 cleavage is a slow process and is not activated by protein kinase C. Pervanadate, a general inhibitor of protein-tyrosine phosphatases, induces a rapid and potent shedding of HER2 ECD. The shedding of HER2 ECD is inhibited by the broad-spectrum metalloprotease inhibitors EDTA, TAPI-2, and batimastat. The tissue inhibitor of metalloproteases-1; an inhibitor of matrix metalloproteases that does not inhibit cleavage by the general protein kinase C-dependent shedding machinery, also inhibited HER2 ECD shedding, whereas tissue inhibitor of metalloproteases-2 did not. These data suggest that HER2 cleavage is a process regulated by an as-yet-unidentified distinct protease.

Topics: Breast Neoplasms; Endopeptidases; Humans; Hydroxamic Acids; Metalloendopeptidases; Phenylalanine; Phosphorylation; Protease Inhibitors; Receptor, ErbB-2; Thiophenes; Tissue Inhibitor of Metalloproteinase-1; Tumor Cells, Cultured; Tyrosine; Vanadates

MCF-7 cells migrate through vitronectin-coated filters in response to insulin-like growth factor I (IGF-I); migration is inhibited by the matrix metalloproteinase (MMP) inhibitor BB-94, but not by the serine proteinase inhibitor aprotinin. MMP-9 was identified in the conditioned medium of MCF-7 cells; in addition, fluorescence-activated cell sorting analysis revealed its presence on the cell surface, where MMP-9 activity was also found using a specific fluorogenic peptide. Furthermore, the messenger RNA encoding MMP-9 was detected in MCF-7 cells by PCR. The IGF-I concentration leading to maximal MCF-7 invasion produces an increase in cell surface proteolytic activity after short incubation periods. At 18 h, however, preincubation of MCF-7 cells with IGF-I produces at 18 h a dose-dependent decrease in cell-associated MMP-9 activity and an increase in soluble MMP-9. MCF-7 invasion is dependent on the alpha(v)beta5 integrin, a vitronectin receptor. The levels of alpha(v)- and beta5-subunits expressed in MCF-7 cells depend on the IGF-I concentration, which triggers an increase in both of these subunits. Based on these results, we suggest that IGF-I-induced MCF-7 cell migration is mediated by the MMP-9 activity on the cell surface and by alpha(v)beta5 integrin.

Topics: Breast Neoplasms; Cell Membrane; Cell Movement; Collagenases; Culture Media, Conditioned; Humans; Insulin-Like Growth Factor I; Integrins; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phenylalanine; Polymerase Chain Reaction; Protease Inhibitors; Receptors, Vitronectin; RNA, Messenger; Thiophenes; Tumor Cells, Cultured

Co-injection of fibroblasts with human epithelial breast-tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor-promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over-production of TIMP-2 was induced in MCF7 cells by in vitro retroviral-mediated gene transfer. TIMP-2-producing MCF7 cells were then co-injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co-inoculated with fibroblasts into nude mice. Both physiological (TIMP-2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor-promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low-molecular-weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Communication; Collagen; Drug Combinations; Fibroblasts; Gene Transfer Techniques; Humans; Laminin; Metalloendopeptidases; Mice; Mice, Nude; Phenylalanine; Protease Inhibitors; Proteoglycans; Stromal Cells; Thiophenes; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

Breast cancer cell lines vary in invasive behavior and one highly invasive cell line (MDA-MB-231) proteolytically degrades extracellular matrix with invadopodia (Thompson et al. 1992, J Cell Physiol, 150, 534-44; Chen et al 1994, Breast Cancer Res Treat, 31, 217-26). Invadopodial proteolysis of extracellular matrix is thought to be necessary for invasion; however, this has not been demonstrated directly. To obtain such evidence, normal (HBL-100) and malignant (MCF-7, MDA-MB-231) breast cells were evaluated for invadopodial proteolysis of extracellular matrix and invasive behavior. We report that invadopodial proteolysis of immobilized fibronectin is positively correlated with invasion of cells into type I collagen gels. Moreover, reducing the proteolytic activity of invadopodia with the metalloproteinase inhibitor, batimastat (BB-94), also decreases invasion indicating that breast cancer cell invasion is dependent upon proteolytically active invadopodia.

Topics: Breast Neoplasms; Collagen; Enzyme Inhibitors; Extracellular Matrix; Female; Fibronectins; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Proteins; Phenylalanine; Pseudopodia; Thiophenes; Tumor Cells, Cultured

Matrix metalloproteinases (MMPs) are involved in the invasion and metastasis of human cancers by mediating the degradation of extracellular matrix components. Therefore, these enzymes constitute promising targets in the development of anticancer therapies. Batimastat ([(4-N-hydroxyamino)-2R-isobutyl-3S-(thienyl-thiomethyl)succinyl]-L- phenyl-alanine-N-methylamide) is one of a new class of agents designed to inhibit MMP activity.. We asked whether batimastat, given as adjuvant therapy after primary tumor resection, could inhibit local-regional tumor regrowth and the formation of lung metastases in a human breast cancer xenograft model. We also explored possible effects of batimastat on breast cancer cell viability and on the accumulation of specific messenger RNAs (mRNAs).. Human MDA-MB-435 breast cancer cells were treated in vitro for 6 days with batimastat at concentrations ranging from 0.1 to 10.0 microM, and then viable cell counts were performed. The activity of collagenases, directly associated with cultured MDA-MB-435 cells or released into their culture fluids, was assessed by gelatin zymography after 1 and 3 days of batimastat treatment (drug range, 0.2-2.0 microM). Athymic nude mice were given daily intraperitoneal injections of batimastat (30 mg/kg body weight) after resection of MDA-MB-435 primary tumors grown in their mammary fat pads; the volumes of tumor regrowths and the numbers and volumes of lung metastases were calculated; neovascularization in the regrowths was assessed by immunohistochemical analysis with an antibody directed against CD31, an endothelial cell antigen. The effect of batimastat treatment on the accumulation of mRNAs encoding specific MMPs and the tissue inhibitor of metalloproteinases-2 (TIMP-2) in cultured cells, primary tumors, and tumor regrowths was measured by RNA dot blotting and hybridization with complementary probes. Linear regression analysis, Student's t tests, and chi-squared analysis were used to evaluate the data.. The viability of cultured MDA-MB-435 cells was not affected by treatment with batimastat; however, measured activities for the 72-kd and 92-kd collagenases released by these cells were reduced after batimastat treatment. Intraperitoneal injection of batimastat significantly inhibited the local-regional regrowth of resected MDA-MB-435 tumors in athymic nude mice (in comparison with control mice, P = .035), and it reduced the incidence (P < .05), number (P = .0001), and total volume (P = .0001) of lung metastases. Batimastat treatment did not affect cellular levels of MMP or TIMP-2 mRNAs.. Batimastat inhibits human breast cancer regrowth and metastasis in a nude mouse xenograft model. Potential mechanisms for batimastat's inhibitory activity do not include direct cell toxicity or alteration of MMP or TIMP mRNA levels.

Topics: Animals; Breast Neoplasms; Chemotherapy, Adjuvant; Chi-Square Distribution; Collagenases; Female; Humans; Linear Models; Lung Neoplasms; Metalloendopeptidases; Mice; Mice, Nude; Neoplasm Recurrence, Local; Neoplasm Transplantation; Phenylalanine; RNA, Messenger; RNA, Neoplasm; Thiophenes; Tumor Cells, Cultured