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Cell migration is a critical determinant of cancer metastasis, and a better understanding of the genes involved will lead to the identification of novel targets aimed at preventing cancer dissemination. KIAA1199 has been shown to be upregulated in human cancers, yet its role in cancer progression was hitherto unknown.Clinical relevance was assessed by examining KIAA1199 expression in human cancer specimens. In vitro and in vivo studies were employed to determine the function of KIAA1199 in cancer progression. Cellular localization of KIAA1199 was microscopically determined. SNAP-tag pull-down assays were used to identify binding partner(s) of KIAA1199. Calcium levels were evaluated using spectrofluorometric and fluorescence resonance energy transfer analyses. Signaling pathways were dissected by Western blotting. Student t test was used to assess differences. All statistical tests were two-sided.KIAA1199 was upregulated in invasive breast cancer specimens and inversely associated with patient survival rate. Silencing of KIAA1199 in MDA-MB-435 cancer cells resulted in a mesenchymal-to-epithelial transition that reduced cell migratory ability in vitro (75% reduction; P.001) and decreased metastasis in vivo (80% reduction; P.001). Gain-of-function assays further demonstrated the role of KIAA1199 in cell migration. KIAA1199-enhanced cell migration required endoplasmic reticulum (ER) localization, where it forms a stable complex with the chaperone binding immunoglobulin protein (BiP). A novel ER-retention motif within KIAA1199 that is required for its ER localization, BiP interaction, and enhanced cell migration was identified. Mechanistically, KIAA1199 was found to mediate ER calcium leakage, and the resultant increase in cytosolic calcium ultimately led to protein kinase C alpha activation and cell migration.KIAA1199 serves as a novel cell migration-promoting gene and plays a critical role in maintaining cancer mesenchymal status.

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of alpha-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metaloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Background, Matrix metalloproteinases (MMPs), especially gelatinase A and gelatinase B (GLB), are believed to be important components of the metastatic process. Tissue Inhibitors of Metalloproteinases (TIMPs) form complexes with MMPs and inhibit cancer dissemination. After local secretion, MMPs and their complexes with TIMPs leach into the blood stream where their concentration can be measured, thereby serving as surrogate markers of disease. Elevated plasma gelatinase B levels have been detected in gastrointestinal cancer and breast cancer. The goal of this study was to determine whether plasma GLB:TIMP complexes also are increased in cancer and whether these tests have potential use as prognostic tumor markers. Methods. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the plasma concentration of GLB:TIMP complexes in patients with cancer. Correlation between ELISA results and clinical outcome was sought. Results. Plasma GLB:TIMP complexes were significantly increased in patients with gastrointestinal cancer and gynecologic cancer, but not in patients with breast cancer. When results from plasma GLB:TIMP complexes and plasma GLB assays were combined (GLB/complexes), abnormal levels of one or both assays were found in 36% and 65% of patients with gastrointestinal and gynecologic cancer, respectively. In Stage IV gastrointestinal cancer, patient survival was shorter (P < 0.001) in the group with increased plasma GLB/complexes than for those with normal plasma levels (4 months vs. 20 months, respectively). Conclusions. The assay of plasma gelatinase B and GLB:TIMP complexes may be clinically useful in predicting survival in subsets of patients with cancer. The possibility of using these assays in early stage cancer to predict metastasis should be studied. Cancer 1995; 76:700-8.

Based upon the hypothesis that metalloproteinases and their inhibitors might be involved in the pathogenesis of Alzheimer's disease, we studied brain samples of eight cases of Alzheimer's disease, six other pathological entities and three elderly controls for tissue inhibitor of metalloproteinase (TIMP) immunoreactivity. Specificity was supported by a loss of immunoreactivity following antigen preabsorption of antisera. Areas studied included ependyma, choroid plexus, frontoparietal, hippocampal and cerebellar cortex, n. basalis of Meynert, basal ganglia, midbrain, pons, and medulla. TIMP positivity was localized to neuritic senile plaques, neurofibrillary tangles and Purkinje cells. The pattern of TIMP plaque staining was similar to that observed with anti tau and SP18 antibodies. It differed from that observed with anti SP40, HAM 56 and GFAP antibodies. The selective localization of TIMP to the neuritic lesions of Alzheimer's disease in a codistribution with the amyloid precursor protein and abnormally phosphorylated and truncated tau supports a possible role for metalloproteinases and their inhibitors in the evolution of these lesions.

Although metastasis accounts for the death of approximately 90% of patients with cancer (1), this field of research was of little interest to most scientists for most of the 20th century. The past decade, however, has witnessed an explosion of interest in metastasis, and the complexity of the process has become mind-boggling. New concepts describing the plethora of mechanisms involved in the metastatic process (eg, premetastatic niche formation, epithelial–mesenchymal transition [EMT], protease-independent cancer cell invasion) have forced us “to think outside the box” (1). In this issue of the Journal, Mochizuki et al. (2) provide novel experimental data explaining how aggressive types of cancer cells are able to avoid apoptosis in the circulatory system at metastatic sites. Prior studies by the authors had demonstrated that increased tumor tissue and serum levels of a metalloproteinase (a disintegrin and metalloproteinase [ADAM]28), whose functions are little known, correlated with poor prognosis and metastasis in patients with breast and lung cancer (3). The authors proposed that ADAM28 contributes to tumor growth by selective digestion of insulinlike growth factor binding protein 3 and connective tissue growth factor (3). However, the molecular mechanisms by which ADAM28 promotes experimental metastases remained elusive. Using a yeast two-hybrid system to screen a human lung cDNA library to identify how ADAM28 promotes the progression of cancer, Mochizuki et al. (2) found that ADAM28 binds to and cleaves von Willebrand's factor (VWF). The surprising aspect of this report is the demonstration that physiological levels of VWF, a prominent plasma protein associated with blood coagulation and thrombosis, induces cancer cell apoptosis. In this study (2), the in vitro experiments demonstrated that rapidly growing and metastatic human carcinoma cell lines expressing high level of ADAM28 were resistant to VWF-induced apoptosis, but apoptosis was induced when the expression or activity of ADAM28 was blocked. Less aggressive cancer cell lines did not express ADAM28 and underwent apoptosis when treated with VWF. The authors demonstrated that VWF-induced cancer cell apoptosis involved the αVβ3 integrin, leading to activation of a mitochondrial cell death pathway. Mochizuki et al. further used a mouse model of lung metastasis and demonstrated that knockdown of ADAM28 expression or activity in cancer cells resulted in diminished metastasis. This antimetastatic effect was associated with enhanced cancer cell apoptosis within blood vessels in the lung and reduced VWF degradation in the blood. The authors concluded that cleavage of proapoptotic VWF by ADAM28 enhances lung metastasis by promoting cancer cell survival within the circulatory system (2). Let us step back in the literature to place the recent findings regarding ADAM28 and VWF (2) into perspective. Scientific investigation of matrix metalloproteinases (MMPs) emerged in the 1980s to explain how cancer cells break out of the confines of a primary tumor to enter (intravasation) and exit (extravasation) from the circulatory system and invade and proliferate at distant metastatic sites (1,4). This enthusiasm, however, was tamped down following the failure of first generation broad-spectrum MMP inhibitors in cancer clinical trials (4). ADAMs have been late comers to the large family of zinc-dependent MMPs. ADAM9, ADAM10, ADAM12, and ADAM17 are intrinsic plasma membrane proteins with established roles in regulating the cancer cell phenotype via ectodomain shedding leading to procancer effects on cell adhesion, migration, proteolysis, signaling and induction of EMT, and ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, and ADAM28 are highly expressed in human malignant tumors (2,3). VWF, a multimeric protein containing binding domains for various proteins, is secreted by endothelial cells and platelets and is well known in primary hemostasis for promoting platelet adhesion to the subendothelium and platelet aggregation (3). VWF also acts as a carrier for coagulation factor VIII. Of relevance to this Editorial, thrombotic thrombocytopenic purpura is a disease involving ADAMTS (with thrombospondin type1 motif)13 and VWF; absence or inhibition of this ADAM results in lack of cleavage of VWF multimers, leading to platelet aggregation and a hypercoagulable state (5). In contrast to ADAM28, which is capable of cleaving both native and denatured VWF, the physiological role of ADAMTS13 is limited to cleavage of denatured VWF, at sites of high shear stress in the arterial circulation. After intravasation, tumor cells have long been known to interact with platelets and the vessel wall, before extravasation from the circulation. VWF has been implicated in the binding of platelet tumor cell thrombi to the endothelium (6,7). Furthermore, human tumor cells increase endothelial cell production of VWF, thereby enhancing adhesion of cancer cells to the endothelium (6). Relevant to the study by Mochizuki et al. (2), an in vitro interaction between VWF and cancer cells involving the Arg-Gly-Asp tripeptide sequence (a binding site for adhesion of the αVβ3 integrin) of VWF and the αVβ3 integrin on the tumor cell surface was previously characterized by Terraube et al. (7). Contrary to the expectation, cell culture studies revealed that VWF did not influence tumor cell proliferation or invasion but instead induced apoptosis (2). This in vitro observation was supported by in vivo experiments demonstrating an increased number of pulmonary metastases in VWF-null mice compared with control mice following intravenous injection of cancer cells. Restoration of VWF plasma levels by injection of VWF inhibited metastasis. These experiments conducted by Mochizuki et al. (2) are consistent with the concept that inactivation of VWF is required in the initial establishment of metastatic foci, independently of its role in hemostasis. Now, how are we to integrate the elegant preclinical studies of Mochizuki et al. (2) to expand our understanding of clinical metastasis? Let us go back to the basics. The mouse pulmonary colonization model used by Mochizuki et al. (2) is considered the “gold standard” in analyzing cancer dissemination. The fact that mouse lung metastasis occurs within weeks, whereas human metastases develop over years, is cause for concern. Furthermore, the extensive proliferation of metastatic cancer cells intravascularly in mouse lungs (8) appears different from pathological examination of human metastases. What other data is needed to “nail down” the role of ADAM28 cleavage of VWF in cancer? An association of VWF levels in blood and tissues and ADAM28 levels in patient tumor tissue with indolent vs aggressive cancers would be revealing. One area of concern deals with an old observation that humans with type O blood group have substantially lower plasma levels of VWF than other ABO groups (9), which, according to Mochzuki et al. (2), might lead to diminished tumor cell apoptosis, hence more aggressive cancers. But, on the contrary, patients with type O blood do not display enhanced tumor aggressiveness (9–11). Other investigators reported increased serum levels of VWF antigen and multimers in patients with cancer, but the issue of correlation of VWF with prognosis is disputed (12). From a wider perspective, how are we to make sense of the enlarging number of genes being implicated in the metastatic process? Will it turn out that individual human tumors require different proteins to complete the metastatic process or will a small panel of “driver genes” be identified? We have the cell and molecular technology, animal models, and clinical tools to solve the problem, but the path to drug discovery for treatment of metastasis and the dream of turning cancer into a “chronic disease” (13) might turn into quite an obstacle.

Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5′-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5′-primer extension study with 5′RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.

Emerging evidence has implicated the role of tumor initiating cells (TICs) in the process of cancer metastasis. The mechanism underlying the conversion of TICs from stationary to invasive remains to be characterized. In this report, we employed less invasive breast cancer TICs, SK-3rd, that displays CD44(high)/CD24(low) with high mammosphere-forming and tumorigenic capacities, to investigate the mechanism by which stationary TICs are converted to invasive TICs. Invasive ability of SK-3rd TICs was markedly enhanced when the cells were cultured under hypoxic conditions. Given the role of membrane type 1-matrix metalloproteinase (MT1-MMP) in cancer invasion/metastasis, we explored a possible involvement of MT1-MMP in hypoxia-induced TIC invasion. Silencing of MT1-MMP by a shRNA approach resulted in diminution of hypoxia-induced cell invasion in vitro and metastasis in vivo. Under hypoxic conditions, MT1-MMP redistributed from cytoplasmic storage pools to the cell surface of TICs, which coincides with the increased cell invasion. In addition, CD44, a cancer stem-like cell marker, inversely correlated with increased cell surface MT1-MMP. Interestingly, cell surface MT1-MMP gradually disappeared when the hypoxia-treated cells were switched to normoxia, suggesting the plasticity of TICs in response to oxygen content. Furthermore, we dissected the pathways leading to upregulated MT1-MMP in cytoplasmic storage pools under normoxic conditions, by demonstrating a cascade involving Twist1-miR10b-HoxD10 leading to enhanced MT1-MMP expression in SK-3rd TICs. These observations suggest that MT1-MMP is a key molecule capable of executing conversion of stationary TICs to invasive TICs under hypoxic conditions and thereby controlling metastasis.

The effects of doxycycline hyclate (DOXY) pleurodesis were compared with those of tetracycline hydrochloride (TCN) in 25 healthy New Zealand White rabbits. One group of rabbits received TCN (pH 2.0) in the left pleural space at a concentration of 35 mg/kg in 3.0 ml of saline solution. Four other groups of rabbits received DOXY in two concentrations (35 mg/kg or 10 mg/kg) and at two pHs (an acid of 2.0 and a NaOH neutralized pH of 7.6). No attempt was made to drain the pleural space after pleurodesis. At autopsy 2 weeks later, bloody fluid filled the pleural cavity and extensive intrapleural adhesions compressed the underlying lung. Microscopically, fibrin and collagen were diffusely deposited in the pleural connective tissue and in the intrapleural adhesions. The visceral pleural thickness increased from its normal value of 0.01 mm to values of 0.5 to 0.9 mm in the TCN and DOXY groups. Fibroblasts were the predominant inflammatory cell in the pleural connective tissue with few neutrophils or lymphocytes. No significant differences of histologic characteristics were observed between the TCN and DOXY groups. Alterations in pH or concentration in the DOXY groups produced similar changes in the gross and microscopic appearance of the pleural space. We conclude that DOXY is as effective as TCN in producing chemical pleurodesis. Doxycycline concentrations of 10 mg/kg were sufficient to substantially sclerose the pleural space in these healthy rabbits. Doxycycline pleurodesis was effective at neutral pH suggesting a mechanism of action independent of acid injury to the mesothelium.

Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.

We hypothesized that inhibition of matrix-degrading metalloproteinases (MMPs) accounts for a portion of the pleural fibrosis and adhesions of tetracycline pleurodesis. MMPs recently have been described in pleural fluid from patients with both exudative and transudative effusions. Since tetracyclines are recognized inhibitors of other metalloproteinases, we investigated their inhibitory capacity in pleural fluid. High concentrations of several different tetracyclines reduced MMP activity of pleural fluid by more than 75 percent. Lower concentrations (≤1 mg/ml) had only modest inhibitory effects. High concentration of of tetracyclines also inhibited cell synthesis of MMPs, in vitro , but other measures of vital cell function were also impaired. We conclude that tetracyclines are effective inhibitors of MMP activity in pleural fluid and may also reduce synthesis of MMPs via non specific cell injury. These data suggest a possible mechanism to account for tetracycline pleurodesis; ie , an inhibition of MMP activity in pleural fluid.

A wealth of knowledge has accumulated over the past four decades on the importance of matrix metalloproteinases (MMPs) in cancer induction, invasion, and metastasis. This chapter aims to provide the reader with recent information to help understand the disconnect between experimental observations implicating the crucial role of MMPs in cancer progression and the historic failure of several broad-spectrum MMP inhibitors in clinical drug trials in advanced cancer. The chemistry and biology of the large MMP family and tissue inhibitors of MMPs (TIMPs) will be summarized. Complexity of MMP function in cancer will be described with an emphasis on pericellular cleavage of extracellular matrix (ECM) and non-ECM substrates. Production of MMPs by stromal cells within a tumor, as well as cancer cells, is well established. Anticancer effects of selected in MMPs are described. The study of cell migration within a three-dimensional collagen matrix has been responsible for broadening our understanding of cancer progression. The involvement of MMPs in the transition from noninvasive to invasive, metastatic cancer, and an emphasis on epithelial-to-mesenchymal transition (EMT) will be presented. New approaches to improve the specificity of MMP inhibitors for use in future clinical trials are discussed.

We identified and characterized type IV collagenase and gelatinase activity in pleural fluid from 32 patients. The capacity to substantially degrade type IV collagen was demonstrated in every pleural sample. Comparable results were also noted for the degradation of a radiolabeled gelatin substrate. Gelatin gel zymography of the pleural fluids revealed two prominent zones of lysis at 66 kDa and 92 kDa. These were identified by specific polyclonal antibodies as human matrix metalloproteinases MMP-2 and MMP-9. The concentration of MMP-2 in pleural fluid, as measured by enzyme-linked immunoassay, averaged 1,622 ng/ml whereas those of MMP-9 were 210 ng/ml. Substrate degradation activity was compared in both serum and pleural fluid from three patients and found to be similar. In serum this enzymatic activity was primarily due to MMP-9 whereas in pleural fluid, the predominant gelatinase was MMP-2. This was confirmed by immunoassay that showed that MMP-2 levels were two to five times higher in pleural fluid than in serum. We conclude that substantial amounts of MMP-2 and, to a lesser degree, MMP-9 are present in pleural effusions. The bioactivity and the immunoactivity of these enzymes did not help to distinguish among pleural fluids characterized as transudates, nonmalignant exudates, or malignant exudates. The differences in the distribution of these enzymes in pleural fluid and blood suggest that their presence is not due simply to the ultrafiltration of plasma, but rather to synthesis by the resident cells at the pleural surfaces.

Numerous studies have reported a correlation between production of 72-kDa (MMP-2) and 92-kDa (MMP-9) type-IV collagenases/gelatinases and the metastatic potential of cancer cells. An abrogating effect of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) on metastases has also been noted. In this report we have used sensitive enzyme-linked immunoassays to measure MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in eight human lung-cancer cell lines which were characterized for biological behavior in nude mice. We demonstrated that the Calu-6 and A549 cell lines with the highest metastatic, invasive and tumorigenic potential secreted the highest levels of MMP-2. MMP-9 and TIMP-1 secretions were comparatively low in all cell lines. TIMP-2 secretion, which exceeded MMP-2 secretion for all cell lines, did not correlate with metastatic potential. To further explore these correlations, the metastatic Calu-6 cell line was transfected with a K-rev-1 cDNA expression construct. The K-rev revertant cell lines demonstrated a more differentiated phenotype and were less tumorigenic, invasive and metastatic in nude mice. Nonetheless, the Calu-6 revertant cell lines secreted higher levels of MMP-2 than the parent cell line. In conclusion, invasion and metastasis by lung-cancer cells requires not only enhanced MMP production, but also other less well-understood tumorigenic characteristics. The multiplicity of factors required by cancer cells for dissemination helps to explain the minute fraction of cancer cells from a primary tumor that ever develop into a metastasis.

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.

Commentary to:A novel and selective membrane type-1 matrix metalloproteinase (MT1-MMP) inhibitor reduces cancer cell motility and tumor growthJuho Suojanen, Tuula Salo, Erkki Koivunen, Timo Sorsa and Emma Pirila

Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we show that HMGA1 is an important oncogene that drives transformation in undifferentiated, large-cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large-cell human lung carcinoma cells. We also show that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target upregulated by HMGA1 in large-cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large-cell lung cancer cells, but not in squamous cell carcinoma cells. In large-cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large-cell carcinoma cells, indicating that the HMGA1–MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large-cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large-cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors. (Mol Cancer Res 2009;7(11):1803–12)

Alpha 2-macroglobulin, a major glycoprotein component of plasma, is unique in its capacity to bind and inhibit the proteolytic activities of all classes of proteinases. Since proteinases implicated in cancer dissemination (type-IV collagenase, plasminogen activator, cathepsins B) are normal constitutents of blood, we have explored the hypothesis that elevated tissue levels of activated proteinases bound to alpha 2M might be detected in plasma of patients with cancer. To test this premise, blood was collected from 149 subjects (33 healthy controls, 31 patients with infections and non-malignant diseases, 16 with myeloproliferative disease, 10 with gastrointestinal cancer, 7 with genito-urinary cancer, 16 with lung cancer, 14 with lymphoma, 11 with miscellaneous cancers and 11 with chronic lymphocytic leukemia and myeloma). Plasma was assayed for alpha 2M-proteinase complexes using a sandwich ELISA which employs a mouse monoclonal antibody (MAb) that binds to a neo-antigenic determinant on complexed alpha 2M and a rabbit polyclonal anti-native human alpha 2M antibody. The concentration of complexed alpha 2M in healthy controls was 14.2 +/- 9.8 micrograms/ml (mean +/- standard deviation). No significant differences in complexed alpha 2M were noted between normal and cancer groups (range 7.4-14.6 micrograms/ml). On the basis of these data, we propose that, in patients with cancer, activated proteinases are bound locally to inhibitors in the tissues and are not available to form complexes with plasma alpha 2M. An alternative explanation is that proteinases are not secreted in excess by cancer cells in vivo.

Circulating tumor cells (CTCs) hold promise for studying advanced prostate cancer. A functional collagen adhesion matrix (CAM) assay was used to enrich CTCs from prostate cancer patients' blood. CAM ingestion and epithelial immuno-staining identified CTCs, which were genotyped using oligonucleotide array comparative genomic hybridization. The highest CTC counts were observed in men with metastatic castration resistant prostate cancer (CRPC) compared to cas- tration sensitive prostate cancer. Copy number profiles for CRPC CTCs were similar to paired solid tumor DNA, and distinct from corresponding DNA from the residual CAM-depleted blood. CAM CTC enrichment may allow cellular and genetic analyses in prostate cancer.

By mining DNA microarray data bases at GenBank™, we identified up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) in human primary and metastatic prostate cancer specimens as compared with nonmalignant prostate tissues. To explore the role of up-regulated MT1-MMP in early stage cancer progression, we have employed a three-dimensional cell culture model. Minimally invasive human prostate cancer cells (LNCaP) were transfected with MT1-green fluorescent protein (GFP) chimeric cDNA as compared with GFP cDNA, and morphologic and phenotypic changes were characterized. GFP-expressing LNCaP cells formed multicellular spheroids with cuboidal-like epithelial morphology, whereas MT1-GFP-expressing cells displayed a fibroblast-like morphology and a scattered growth pattern in type I collagen gels. Cell morphologic changes were accompanied by decreased epithelial markers and enhanced mesenchymal markers, consistent with epithelial-to-mesenchymal transition. MT1-MMP-induced morphologic change and cell scattering were abrogated by target inhibition of either the catalytic domain or the hemopexin domain. We further demonstrated that MT1-MMP-induced phenotypic changes were dependent upon up-regulation of Wnt5a, which has been implicated in epithelial-to-mesenchymal transition. We conclude that MT1-MMP plays an important role in early cancer dissemination by converting epithelial cells to migratory mesenchymal-like cells.

Numerous studies have shown that intact cancer cells and cell extracts have the capacity to lyse erythrocytes in vitro. The transformation of NIH-3T3 fibroblasts by ras oncogenes has recently been demonstrated to result in tumour cells releasing a haemolytic factor. The purpose of this study has been to purify and further characterise the soluble tumour haemolytic factor (sTHF) produced by mouse fibroblasts transformed by T24 human bladder cancer DNA and by the cloned Harvey murine sarcoma viral oncogene. To this end, transformed fibroblasts were cultivated in serum-free medium. The cell-free supernatant was treated with ammonium sulphate and the precipitate achieved at 60-100% saturation was dialysed and applied to a gel filtration column. A haemolytic factor was eluted with an Mr between 65,000 and 75,000. Zinc chelate and strong anion exchange column chromatography resulted in greater than 3,000-fold enrichment of sTHF. SDS-PAGE of sTHF resulted in a single protein band of 66,000 Da. Soluble THF had no immunological cross-reactivity with known cytokines produced by lymphocytes and macrophages. The pathophysiological role of sTHF in cancer remains to be determined. Images Figure 3

Tumor proteinases are considered to be important in the process of cancer invasion and metastasis. We have proposed that the surface membrane localization of these proteinases places them in an optimal site to facilitate the invasion of surrounding extracellular matrix. In this study, we have used the organic solvent, n-butanol, and the detergent, n-octyl-glucoside, to sequentially extract metalloproteinases from crude plasma membranes of human RWP-I pancreatic cancer cells. Anion exchange chromatography and gel permeation chromatography were employed to further purify enzymes with the capacity to degrade gelatin, type-IV collagen, and carboxymethylated transferrin. Gelatin zymography was used to demonstrate proteinase bands of 92, 70 and 62-kDa. Immunoblotting of solubilized, partially purified pancreatic cancer plasma membrane proteins using polyclonal rabbit antibodies, which have specificity for type-IV collagenase/gelatinase, resulted in the recognition of a 70-kDa protein, but not the 92-kDa gelatinase. A type-IV collagenase/gelatinase of 68-kDa was similarly identified in A2058 human melanoma cancer cell plasma membranes.

Long-term chlorpromazine therapy has been associated with the asymptomatic development of a high incidence of antinuclear antibodies, coagulation inhibitors, and increased serum levels of IgM. The purpose of this study has been to characterize the natural history of this chlorpromazine-induced (CPZ) immunopathy. To this end we carried out a prospective study of schizophrenic patients with the immunopathy to compare the effect of continuing CPZ versus switching to haloperidol therapy. Although no marked differences were noted between the 2 groups at the end of 5 years, 6 of 29 patients who continued to receive CPZ, as compared to none of 14 patients on haloperidol, had progressive elevations of serum IgM. In spite of a high incidence of antinuclear antibodies, none of the patients developed a lupus-like syndrome. One patient, however, who had been maintained on CPZ for more than 15 years, developed Waldenstrom macroglobulinemia, as characterized by an IgM monoclonal gammopathy and a lymphocyte immunoglobulin heavy and kappa light chain gene rearrangement. Another CPZ-treated patient developed immune thrombocytopenia. Based on the potential serious sequelae of prolonged stimulation of the immune system by CPZ, we recommend that patients who develop an increase in serum IgM while on CPZ be switched to other types of anti-psychotic medications.

We assessed the association of genetic variation in MMP3 and MMP9 with risk of myocardial infarction and stroke.A case-control study was conducted among members of Group Health (GH), a large-integrated health care delivery system. Case subjects with incident non-fatal myocardial infarction (n=854), ischemic stroke (n=367), and hemorrhagic stroke (n=66) were identified and validated. A matched control group was selected from among GH members without myocardial infarction or stroke (n=2696). Haplotype-tagging sets of single-nucleotide polymorphisms (SNPs) in MMP3 and MMP9 were genotyped.MMP3 haplotype 2 was associated with reduced risk of myocardial infarction (adjusted odds ratio (OR) per copy=0.80, 95% confidence interval 0.66, 0.98) and increased risk of hemorrhagic stroke (OR=1.69, 95% confidence interval 1.05, 2.75). Results for MMP3 haplotype 2 and ischemic stroke resembled those for myocardial infarction but did not achieve statistical significance (OR=0.85, 95% confidence interval 0.64, 1.12). No individual SNP identified MMP3 haplotype 2, and none of the individual MMP3 SNPs were associated with myocardial infarction or stroke. MMP9 haplotypes or SNPs were not associated with myocardial infarction or stroke.MMP3 haplotype may predict both cardiac events and stroke.

Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1α (HIF-1α) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1α/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1α, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD.

Scientific exploration of cancer in the past century has primarily focused on the concept of uncontrolled growth of cancer cells with little attention to the non-neoplastic component of tumors. More recent studies brought to light the important positive and negative effects of the local host response to cancer. The tumor microenvironment is a specialized niche that provides the survival signals necessary for cancer cells to undergo proliferation, neovascularization, invasion, and metastasis. The tumor microenvironment thus provides the fertile soil for tumor growth and the selective survival advantages necessary for dissemination to other organs (Seed and Soil Hypothesis) (Fidler 2002; Paget 1889). Beginning in the early stages and continuing throughout tumor development, cancer cells secrete multiple cytokines, growth factors, and proteases to re-shape their surrounding connective tissue. This leads to the formation of a ‘reactive’ stroma that supports tumor progression by inducing reactions such as angiogenesis and inflammation. Stromal cells, such as fibroblasts, leukocytes, and endothelial cells, become activated and release additional growth factors and proteases to foster tumor growth. The extracellular matrix (ECM), another component of tumor stroma, acts as a scaffold for structural support, as well as a specialized reservoir of macromolecules, proteases and their inhibitors, growth factors, and cytokines. By inducing compositional changes in the ECM, cancer cells regulate and modify the contextual information needed for cell proliferation, migration, and invasion, thereby controlling their own fate. The reciprocal (paracrine)

Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein α are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH2 terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices. (Cancer Res 2006; 66(9); 4652-61)

The relationship between loss of intercellular adhesion and the biologic properties of human squamous cell carcinoma is not well understood. We investigated how abrogation of E-cadherin-mediated adhesion influenced the behavior and phenotype of squamous cell carcinoma in 3D human tissues. Cell-cell adhesion was disrupted in early-stage epithelial tumor cells (HaCaT-II-4) through expression of a dominant-negative form of E-cadherin (H-2Kd-Ecad). Three-dimensional human tissue constructs harboring either H-2Kd-Ecad-expressing or control II-4 cells (pBabe, H-2Kd-EcadDeltaC25) were cultured at an air-liquid interface for 8 days and transplanted to nude mice; tumor phenotype was analyzed 2 days and 2 and 4 weeks later. H-2Kd-Ecad-expressing tumors demonstrated a switch to a high-grade aggressive tumor phenotype characterized by poorly differentiated tumor cells that infiltrated throughout the stroma. This high-grade carcinoma revealed elevated cell proliferation in a random pattern, loss of keratin 1 and diffuse deposition of laminin 5 gamma2 chain. When II-4 cell variants were seeded into type I collagen gels as an in vitro assay for cell migration, we found that only E-cadherin-deficient cells detached, migrated as single cells and expressed N-cadherin. Function-blocking studies demonstrated that this migration was matrix metalloproteinase-dependent, as GM-6001 and TIMP-2, but not TIMP-1, could block migration. Gene expression profiles revealed that E-cadherin-deficient II-4 cells demonstrated increased expression of proteases and cell-cell and cell-matrix proteins. These findings showed that loss of E-cadherin-mediated adhesion plays a causal role in the transition from low- to high-grade squamous cell carcinomas and that the absence of E-cadherin is an important prognostic marker in the progression of this disease.

Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

SummaryMatrix metalloproteinases (MMPs) play an important role in many biological and pathological processes including tissue remodeling, wound healing, inflammation, atherosclerosis, and cancer. Numerous publications have supported the concept that activated MMP-2 enhances agonist-induced platelet aggregation and activated MMP-9 inhibits platelet aggregation. In this study, we demonstrated that the organomercurial compound, 4-aminophenyl mercuric acetate (APMA), which is routinely employed to activate latent MMPs at a concentration of 1000 μ M, induces platelet aggregation at low concentration (5 μ M) and inhibits agonist-induced platelet aggregation at concentrations ≥ 50 μ M. Activated MMP-2, MMP-1, and MMP-9, following removal of APMA by ultrafiltration through an anisotropic membrane, exert no independent effect on platelet aggregation. Acetylsalicylic acid and BAPTA inhibited APMA-induced platelet aggregation indicating that the APMA mediated pathway of platelet activation is dependent upon thromboxane and calcium signaling. Zinc chelation with 1,10-phenanthroline, which inhibits zincdependent proteins including metalloproteinases, also abrogated platelet functional responses to APMA. Additional studies will be required to clarify the mechanism of the biphasic effect of APMA on platelet aggregation.

Purpose To determine whether a matrix metalloproteinase inhibitor improves progression-free survival (PFS) in patients with metastatic breast cancer who have responding or stable disease after first-line chemotherapy. Patients and Methods One hundred seventy-nine eligible patients were randomly assigned to receive oral marimastat (10 mg bid; n = 114) or a placebo (n = 65) within 3 to 6 weeks of completing six to eight cycles of first-line doxorubicin- and/or taxane-containing chemotherapy for metastatic disease. Patients were evaluated every 3 months until disease progression. Results When comparing placebo with marimastat, there was no significant difference in PFS (median, 3.1 months v 4.7 months, respectively; hazard ratio, 1.26; 95% CI, 0.91 to 1.74; P = .16) or overall survival (median, 26.6 months v 24.7 months, respectively; hazard ratio, 1.03; 95% CI, 0.73 to 1.46; P = .86). Patients treated with marimastat were more likely to develop grade 2 or 3 musculoskeletal toxicity (MST), a known complication of the drug indicative of achieving a biologic effect, compared with patients administered placebo (63% v 22%, respectively; P < .0001). Patients with grade 2 or 3 MST had significantly inferior survival compared with patients who had grade 0 or 1 MST (median, 22.5 months v 28.2 months; P = .04). In addition, patients who had a marimastat plasma concentration of at least 10 ng/mL at month 1 and/or 3 were significantly more likely to have grade 2 to 3 MST (P < .0001). Conclusion Marimastat does not prolong PFS when used after first-line chemotherapy for metastatic breast cancer. Patients with higher marimastat levels exhibited MST, and MST was associated with inferior survival.

Over the past three decades the Veterans Affairs (VA) Research program has evolved into a powerful, peer-reviewed funding mechanism for basic and translational research that has resulted in numerous important contributions to medical science and improvements in patient care. Continuity in VA Merit Review funding has fostered and nurtured the scientific careers of a large number of physician-scientists who have remained devoted to the mission of performing creative and innovative research that affects the patient care mission of the VA. VA medical research policies have undergone a major overhaul in the past year. Although many of these changes (de-emphasizing bench research and revamping the peer review process) have recently been reversed, the future direction of VA research remains in flux. The goal of this manuscript is to demonstrate the importance of the Merit Review medical research funding mechanism not just to the VA, but to the entire nation's health care system. To achieve this goal, the opinions of 65 established VA medical investigators were obtained regarding the past success and future direction of VA research. The conclusions reached include the following. 1) Merit Review research funding has been essential to the training, recruitment, and retention of productive VA physician-scientists. 2) The VA research program has contributed both basic and clinical innovations that have led to improvements in medical care. Contributions of VA researchers to excellence in many aspects of patient care at VA hospitals have been extraordinary. 3) Development of initiatives that entice outstanding Ph.D.'s to develop their careers in the VA has been crucial to the success of the program. 4) The VA research program has fostered a mutually beneficial relationship with affiliated medical schools. 5) Better methods to quantify VA research contributions and outcomes are essential for future program development.

Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities.We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion.Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels.Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

The chemokine CXCL12 (SDF-1) and its receptor, CXCR4, have been implicated in organ-specific metastases of several malignancies. Head and neck squamous cell carcinoma (HNSCC) predominantly metastasizes to lymph nodes, and recent evidence has shown that CXCL12 stimulates HNSCC migration. We explored the potential role of CXCR4 in mediating other metastatic processes in HNSCC cells. CXCR4 mRNA and cell-surface expression was assessed in HNSCC cell lines. CXCR4 mRNA expression was detected in five HNSCC cell lines. Cell-surface CXCR4 was also detected in each of the HNSCC cell lines and in resected HNSCC tissues. CXCL12 induced rapid intracellular calcium mobilization in a metastatic HNSCC cell line (HN), as well as rapid phosphorylation of ERK-1/2. HNSCC cell adhesion to fibronectin and collagen was increased by CXCL12 treatment, while the addition of an inhibitor of ERK-1/2 signaling, PD98059, reduced the effects of CXCL12. CXCL12 also increased the active matrix metalloproteinase (MMP)-9 secreted. Thus, HNSCC cells express functional CXCR4 receptors that induce rapid intracellular signaling upon binding to CXCL12. Such binding leads to increased HNSCC cell adhesion and MMP secretion, suggesting that CXCR4 may be a novel regulator of HNSCC metastatic processes.

Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules;15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.

Publisher Summary Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMPs) are involved in numerous physiologic and pathologic processes. Considerable interest has evolved over the past decade in the measurement of MMPs and TIMPs in blood and urine as an aid in the diagnosis and prognosis of disease. The goal of this chapter is to provide a comprehensive review of the subject with the focus on potential future developments and applications of the technology. The chapter also discusses involvement of MMPs and TIMPs in pathophysiology of disease such as cancer, inflammatory diseases, liver disease, cardiovascular disease, lung diseases, central nervous system disease, shock syndromes, chronic wounds and inflammation of the skin, and oral cavity. Several assays for measurement of MMPs and TIMPs in body fluids such as pitfalls in measurement and origin of MMPs and TIMPs in blood are also discussed in this chapter. Based on research implicating MMPs in various pathologic processes, this chapter has described the measurement of MMPs and TIMPs in the blood and urine of patients with various diseases. Another consideration is whether measurement of blood and urinary levels of MMPs and TIMPs have potential utility in general screening for disease in periodic health examinations. In this chapter, comparisons to baseline MMP measurements for single individuals might be useful in detecting development of disease over protracted periods of time, for example, progressive development of cardiovascular disease.

Purpose Does the release of plasma matrix metalloproteinase-9 (MMP-9) by radiation-activated airway epithelial cells and infiltrating inflammatory cells play a role in the radiation damage or repair process in the lungs? We evaluated lung damage by ionizing radiation using plasma levels of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and MMP-3 as biologic markers of tissue damage, and also their relationship to changes in pulmonary epithelial permeability, clinical signs and symptoms, and lung structural changes. Methods and materials Seven serial studies were conducted in each of 8 patients undergoing chest radiotherapy (RT) for lung or breast cancer, beginning before the first treatment (baseline) and then biweekly to ∼100 days during and after RT. Chest radiographs were monitored for each patient. Sandwich enzyme-linked immunoassays (ELISA) were used to measure plasma MMP-3, MMP-9, and TIMP-1 levels. Lung permeability was evaluated by measuring the rate of epithelial clearance of ∼150 μCi (∼5.6 MBq) inhaled 99m Tc diethylenetriamine pentaacetate aerosol (DTPA). Results Lung and breast cancer resulted in very high plasma levels of MMP-9 (126–893 ng/mL) and TIMP-1 (496–8,985 ng/mL) in all subjects studied before initiation of RT. This compares with plasma MMP-9 and TIMP-1 values in healthy volunteers of 29 ± 11 ng/mL and 436 ± 86 ng/mL, respectively. RT was followed by a sharp decrease in plasma MMP-9 within the first 2 weeks, but without a corresponding change in TIMP-1. In contrast, plasma MMP-3 levels, which are generally increased with inflammation, were elevated in only 1 of 5 subjects. Conclusion Lung and breast cancer are associated with high plasma levels of MMP-9 and TIMP-1. These high baseline plasma levels of MMP-9 were reduced in the first 2 weeks of RT in 7 of 8 subjects, and TIMP-1 plasma levels remained high in all subjects. The decrease in plasma MMP-9 after initiation of chest RT appears to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiation-induced lung damage. Likewise, the lack of a rise in plasma MMP-3 levels does not support a role for MMP-3 in tissue injury or repair in the lung. It remains to be determined whether plasma MMP-9 measurements will serve as a useful parameter in predicting cancer relapse.

The decade of the 1990s was ripe with enthusiasm for the use of MMPIs to treat cancer. Limitations to new cytotoxic chemotherapy approaches to treat solid cancers and a better understanding of tumor biology provided a strong impetus for alternative drug development. It is estimated that the pharmaceutical industry invested at least a billion dollars in this effort. Because MMPIs represent an entirely different therapeutic modality from proven anti-cancer agents, many of the therapeutic trials designed to test MMPIs in human patients with cancer bypassed traditional approaches to evaluate drug efficiency. The concept of systematic progression from small phase I (dose escalation to toxicity to examine drug safety), to phase II (drug treatment of patients with cancer types considered to be good candidates for the selected drug), to phase III (randomized trial of new drug versus best available therapy to determine drug efficacy) trials was modified. Much to the chagrin of everyone involved in these studies, the randomized trials of MMPIs in advanced cancer have, pretty much, flopped. This review article will attempt to dissect out aspects of previous human and animal studies that may be helpful in making decisions about the future of MMPI drug development for the treatment of cancer. The important questions to be addressed in this report are: What are the lessons that we have learned from preclinical (animal models) and clinical studies of MMPIs in cancer? Are we ready to abandon MMPIs as a therapeutic modality in cancer (termination of phase III trials) or do we need to have a better understanding of the myriad effects of MMPs in cancer before we proceed to develop different types of drugs that alter MMP activity in patients with cancer (beginning of new phase I trials)?

Publisher Summary Matrix metalloproteinases (MMPs) belong to the family of zinc endopeptidases collectively referred to as metzincins. The metzincin superfamily is distinguished by a highly conserved motif containing three histidines that bind zinc at the catalytic site and a conserved methionine that sits beneath the active site. The metzincins are subdivided into four multigene families: serralysins, astacins, ADAMs/adamalysins, and MMPs. Once activated, MMPs degrade a variety of extracellular matrix components and assorted other proteins including growth factors, growth factor binding proteins, and protease inhibitors. The ability to degrade extracellular matrix proteins is essential for any cell to interact properly with its immediate surroundings and for multicellular organisms to develop and function normally. MMPs also generate matrix protein fragments, which have functional activity of their own. These various functions of MMPs modulate cell invasion and metastasis, cell migration, apoptosis, and angiogenesis. The belief that an MMP is involved in a given biologic/pathologic process is often based on the strength of its association with that process and the existence of plausible mechanisms that can be tested by experimental approaches. Because of extensive redundancy of MMP function in animals, it has been difficult to appreciate the role of individual genes in various experimental models. The multiplicity of MMPs with distinct but somewhat overlapping functions appears to act as a safeguard against loss of regulatory control. The known MMP genes have been divided into four subfamilies based on gene structure. Group I consists of the collagenase subfamily. Group 2 consists of the gelatinases/modular alteration (fimodular a-like domains) subfamily. Group 3 consists of the variant hemopexin exon subfamily. Group 4 consists of the variant catalytic exon and unique 3′-end exon (plasma membrane and cytoplasmic domains) subfamily, which consists of the membrane type-matrix metalloproteinases (MT-MMPs).

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17–18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.

Cancer progression is a multistage process in which cancer cells undergo genetic changes that lead to phenotypic alterations, including the capacity to metastasize. An overwhelming amount of experimental evidence has been accumulated to implicate matrix metalloproteinases (MMPs) and plasminogen activators (PAs) in invasion and metastasis. However, specific MMPs appear to have different functions depending on the experimental model and the stage of cancer. The fact that most MMPs and PAs in human cancers are produced by reactive stromal cells, such as fibroblasts, endothelial cells, and inflammatory cells surrounding the tumor rather than cancer cells themselves needs to be better understood. Proteases may be good anticancer targets in that they appear to be required for the stromal tissue remodeling associated with angiogenesis and tumor metastasis. However, true clinical efficacy may require combination therapy with cytotoxic agents, as well as long-term administration, possibly at earlier stages of cancer. Numerous highly effective hydroxamic acid-based MMP inhibitors (MMPIs) have been developed, three of which are in clinical trials on patients with advanced cancer. An exciting new approach in the development of more specific MMPIs involves the use of phage display random peptide libraries to isolate selective enzyme inhibitors. The prototype synthetic peptide inhibited the migration of human endothelial cells and tumor cells, prevented tumor growth and invasion in animal models, targeted angiogenic blood vessels in vivo, and improved survival of mice bearing human tumors.

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels.

Introduction: The chronic Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal stem cell disorders characterized by unregulated stem cell expansion and overproduction of blood cells. The acquired mutation JAK2V617F plays a central role in the pathogenesis of MPN. Ruxolitinib was the first FDA-approved JAK2 inhibitor for the treatment of MPNs; however, its use is limited by various toxicities due to its suppression of normal marrow function. Because its toxicity is related to dosage, combinations of drugs may enhance the therapeutic effectiveness of Ruxolitinib and permit the administration of lower doses of Ruxolitinib. Cancer cells depend on a continued supply of glucose and glutamine for cell survival and proliferation. Glutaminase (GLS) is the key enzyme in glutamine metabolism. We report here that JAK2V617F mutation is associated with increased cellular metabolism and the GLS inhibitor BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide) improves the therapeutic efficacy of Ruxolitinib in both JAK2V617F-mutant cell lines and MPN patient samples. Methods: The JAK2V617F-mutant human erythroleukemia (HEL) cell line and the murine prolymphoid cell line Ba/F3 expressing wild-type or mutant JAK2 (BaF3-hEPOR-JAK2wt cell and BaF3-hEPOR-JAK2V617F cell) were maintained as previously described. Cells were treated with DMSO, BPTES (Sigma¨), and/or Ruxolitinib (Selleckchem¨) and cell proliferation was assessed by counting bright live cells in a hemocytometer using the trypan blue exclusion method. Mitochondrial respiration (oxygen consumption rate, OCR) and glycolysis (extracelluar acidification rate, ECAR) were measured using a Seahorse¨ XFe96 Bioanalyzer. We used 13C stable isotope analysis to determine differences in glucose and glutamine utilization. BaF3-hEPOR-JAK2wt cell and BaF3-hEPOR-JAK2V617F cell were grown in [U6]-13C glucose or [U5]-13C glutamine for 24 hours and metabolites extracted and prepared for GC-MS analysis. Data were analyzed using MassHunter software (Agilent Technologies¨). MPN patient specimens were obtained with informed consent and approval by the IRB of Northport VA Medical Center. Peripheral blood CD34+ cells from MPN patients were isolated using immunomagnetic beads (Miltenyi¨) as we previously did. MPN CD34+ cells were treated with DMSO, BPTES, and/or Ruxolitinib and assayed in methylcellulose semisolid medium (Stem Cell Technologies¨) with growth factors. Colonies were counted after 14 days of incubation. Results: OCR and ECAR were increased by 55% (p < 0.0001) and 2-fold (p < 0.0001), respectively, in BaF3-hEPOR-JAK2V617F cells compared to BaF3-hEPOR-JAK2wt cells. This indicated that the JAK2V617F-mutant cells have higher metabolic activity than JAK2WT cells. Stable isotope analysis using GC/MS showed that JAK2V617F-mutant cells had increased utilization of glucose and glutamine into the TCA cycle as well as increased lactate production from glucose. There was a higher glutaminase activity in the JAK2V617F-mutant cells than the JAK2WT cells. Next we determined the effect of the glutaminase inhibitor, BPTES, on Ruxolitinib-mediated inhibition of cell proliferation in JAK2V617F-mutant cell lines. While BPTES (5µM) had little effect on HEL cell growth, the combination of BPTES (5µM) and Ruxolitinib (1.5µM) significantly inhibited HEL cell growth by 66% while Ruxolitinib alone inhibited cell growth by only 35% (p = 0.04). Similar results were obtained with the BaF3-hEPOR-JAK2V617F cells. (Figure 1) Finally, we tested the combination therapy of BPTES and Ruxolitinib in peripheral blood CD34+ cells from 4 MPN patients (2 PMF and 2 ET). The combination of BPTES (2uM) and Ruxolitinib (125nM) suppressed total hematopoietic colony growth of MPN CD34+ cells by 91% in comparison to 68% inhibition by Ruxolitinib alone (p = 0.007). Both CFU-GM colony and BFUe colony were suppressed by the combination therapy more than by Ruxolitinib alone although it was only significant for CFU-GM colony formation. (Figure 2) Conclusion: Our study showed that JAK2V617F mutation was associated with increased cellular metabolism. Importantly, a glutaminase inhibitor enhanced the therapeutic efficacy of Ruxolitinib in both JAK2V617F-mutant cell lines and MPN patient peripheral blood CD34+ cells. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Purpose: Cancer antigen 125 (CA125) is recognized as the leading biomarker in monitoring Epithelial Ovarian Cancer (EOC). More recently, invasive circulating tumor cells (iCTCs) have been introduced as a biomarker for metastatic potential. The goal of this research is to compare this biomarker with CA125 to determine the response to surgery and chemotherapy. Experimental Design: We compared clinical parameters with serial assays for iCTCs and CA125 in 129 fresh blood and 169 serum samples, respectively, from 31 patients who were receiving surgery and chemotherapy. iCTCs were capable of ingesting matrices (Cell Adhesion Matrix CAM+) and identified using positive tumor markers, Epi (EPCAM and CA125 or seprase and CD44), and negative selection markers, which exclude Epi-positive hematopoietic lineage (HL) cells, and enumerated by flow cytometry. iCTCs and CA125 levels were measured at time point less than 3-days apart. Results: A significant correlation was identified between changes in iCTC counts and CA125 levels (r=0.67, P4 serial samples during therapy, changes in iCTC counts provided earlier measure of therapy response than CA125. Conclusion: This analysis showed that iCTC assay compared favorably with CA125 to monitor the response of chemotherapy in EOC patients, whereas changes of CA125 correlated better with reduced tumor burden after surgery. Citation Format: Wen-Tien Chen, Huan Dong, Qiang Zhao, Jie Yang, QIAO ZHANG, Stanley Zucker, Michael L. Pearl. Prognostic analysis of invasive circulating tumor cells (iCTCs) to monitor epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2014-LB-180

Mechanical ventilation has become an indispensable therapeutic modality for patients with respiratory failure. However, a serious potential complication of MV is the newly recognized ventilator-induced acute lung injury. There is strong evidence suggesting that matrix metalloproteinases (MMPs) play an important role in the development of acute lung injury. Another factor to be considered is extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN is responsible for inducing fibroblasts to produce/secrete MMPs. In this report we sought to determine: (1) the role played by MMPs and EMMPRIN in the development of ventilator-induced lung injury (VILI) in an in vivo rat model of high volume ventilation; and (2) whether the synthetic MMP inhibitor Prinomastat (AG3340) could prevent this type of lung injury. We have demonstrated that high volume ventilation caused acute lung injury. This was accompanied by an upregulation of gelatinase A, gelatinase B, MT1-MMP, and EMMPRIN mRNA demonstrated by in situ hybridization. Pretreatment with the MMP inhibitor Prinomastat attenuated the lung injury caused by high volume ventilation. Our results suggest that MMPs play an important role in the development of VILI in rat lungs and that the MMP-inhibitor Prinomastat is effective in attenuating this type of lung injury.

This postdoctoral research training grant entitled "examination of the role of membrane type 1-matrix metalloproteinase (MT1-MMP) in breast cancer metastasis" was initiated in August 1998 and terminated in July 2001. This grant provided Dr. Cao with a great opportunity to be trained to fight the war against breast cancer. Reviewing the progress of the past three years, Dr. Cao reached the goals that he originally proposed. His work during the last three years was highly recognized in the field as demonstrated by several awards including a Young Investigator award and Scholar-in-Training award from American Association for Cancer Research (AACR) and by his publications. During the training period, Dr. Cao was promoted to a Research Assistant Professor which fulfills the goal that Dr. Cao initially proposed of becoming an independent scientist. Dr. Cao is very appreciative of the US Army Medical Research and Material Command committee for providing him with such a training opportunity in a highly competitive field.

Experimental studies performed prior to 1990 led to the widely held belief that matrix metalloproteinases (MMPs) produced by cancer cells are of critical importance in tumor invasion and metastasis. Based on this evidence, the pharmaceutical industry produced several well tolerated, orally active MMP inhibitors (MMPIs) which demonstrated efficacy in mouse cancer models. Phase III clinical trials initiated in 1997-98 using marimastat, prinomastat (AG3340), and BAY 12-9566 alone or in combination with standard chemotherapy in patients with advanced cancers (lung, prostate, pancreas, brain, GI tract) have recently been reported; no clinical efficacy was demonstrated. Bayer and Agouron have discontinued their ongoing Phase III drug trials of MMPIs in advanced cancer. In retrospect, the failure of MMPIs to alter disease progression in metastatic cancer might have been anticipated since MMPs appear to be important in early aspects of cancer progression (local invasion and micrometastasis) and may no longer be required once metastases have been established. Our understanding of MMP pathophysiology in cancer has expanded considerably in the past 10 years. Current views indicate that: (1) most MMPs in tumors are made by stromal cells, not carcinoma cells; (2) cancer cells induce stromal cells to synthesize MMPs using extracellular matrix metalloproteinase inducer (EMMPRIN) and cytokine stimulatory mechanisms; and (3) MMPs promote cell migration and the release of growth factors sequestered in the extracellular matrix. MMPs have a dual function in tumor angiogenesis: MMP-2 and MT1-MMP are required in breaking down basement membrane barriers in the early stage of angiogenesis, while other MMPs are involved in the generation of an angiogenic inhibitor, angiostatin. In spite of considerable recent progress in identifying multiple roles of MMPs in disease, our understanding of MMP function in cancer is far from complete (see Table 1). Based on accumulated data, it is recommended that future MMPI trials focus on: (1) patients with early stage cancer; (2) the use of MMPIs along with chemotherapy; (3) the measurement of MMPs in tumor tissue and blood as a means of identifying patients who are more likely to respond to MMPI therapy; and (4) identification of biomarkers that reflect activation or inhibition of MMPs in vivo.

Tetracyclines (TCs) and their non-antimicrobial analogs (CMTs) have therapeutic potential to inhibit tissue destructive disease processes, such as cancer invasion and metastasis, by inhibiting certain matrix metalloproteinases. Enhanced matrix metalloproteinase-2 (MMP-2; gelatinase A) activity has been correlated to cancer invasiveness, and membrane type MMP (MT1-MMP) expressed by tumor cells is involved in localizing and activating pro-MMP-2, a pathway believed to mediate cancer induced tissue breakdown. CMT-3 (6-demethyl, 6-deoxy, 4-dedimethylamino TC) has been shown to experimentally suppress prostate cancer, colon adenocarcinoma and melanoma invasiveness in cell culture and to inhibit tumor growth and metastasis in vivo and was used in the current in vitro study. Confluent MT1-MMP transfected COS-1 cells were harvested, washed thoroughly, subjected to N(2) cavitation and cell membrane enriched fractions were isolated by sequential centrifugations. This MT1-MMP preparation exhibited (i) pro-MMP-2 activating activity as shown by molecular weight shift of this gelatinase from 72 kDa to 62 kDa using gelatin zymography, and (ii) the ability to degrade both [(3)H-methyl] gelatin and casein at 37 degrees C. Adding CMT-3 at final concentrations of 5--20microM inhibited MT1-MMP gelatinolytic and caseinolytic activity, blocked MT1-MMP activation of pro-MMP-2, and decreased invasiveness (using the Matrigel system) of HT-1080 fibrosarcoma cells. The inhibition of MT1-MMP by CMT-3 may partially explain the inhibition of cancer cell -mediated tissue breakdown and invasiveness by this non-antimicrobial tetracycline analog.