Biomedical subjects

Barbara Fingleton

Publications and source records attributed to Barbara Fingleton.

18 recordsLinked to original sources

PubMed2006

Analysis of host- and tumor-derived proteinases using a custom dual species microarray reveals a protective role for stromal matrix metalloproteinase-12 in non-small cell lung cancer.

We used a customized Affymetrix protease microarray (Hu/Mu ProtIn chip) designed to distinguish human and mouse genes to analyze the expression of proteases and protease inhibitors in lung cancer. Using an orthotopic lung cancer model, we showed that murine matrix metalloproteinase (MMP)-12, MMP-13, and cathepsin K were up-regulated in tumor tissue compared with normal mouse lung. To determine the relevance of stromal proteases detected using this model system, we compared the results to an analysis of human lung adenocarcinoma specimens using the U133 Plus 2.0 Affymetrix microarray. MMP-12, MMP-13, and cathepsin K showed an increase in expression in human tumors compared with normal lung similar to that seen in the orthotopic model. Immunohistochemical analysis confirmed MMP-12 expression in the stroma of human lung tumor samples. To determine the biological relevance of stromal MMP-12, murine Lewis lung carcinoma cells were injected into the tail vein of syngeneic wild-type (WT) and MMP-12-null mice. MMP-12-null and WT mice developed equivalent numbers of lung tumors; however, there was a 2-fold increase in the number of tumors that reached >2 mm in diameter in MMP-12-null mice compared with WT controls. The increase in tumor size correlated with an increase in CD31-positive blood vessels and a decrease in circulating levels of the K1-K4 species of angiostatin. These results show a protective role for stromal MMP-12 in lung tumor growth. The use of the Hu/Mu ProtIn chip allows us to distinguish tumor- and host-derived proteases and guides the further analysis of the significance of these genes in tumor progression.

Animals↗

PubMed2006

Matrix metalloproteinase-9 from bone marrow-derived cells contributes to survival but not growth of tumor cells in the lung microenvironment.

The role of specific stromal-derived matrix metalloproteinases (MMPs) was analyzed in experimental metastasis assays in wild-type and either MMP-9, MMP-7, or MMP-2 null mice. MMP-9 null mice showed an 81% reduction in Lewis lung carcinoma tumor number, whereas MMP-7 null mice showed a 42% increase in tumor number, and there was no difference in tumor number in MMP-2 null mice compared with wild-type controls. Similarly, in an orthotopic model of lung cancer, 50% fewer MMP-9 null mice were able to establish tumors in the lung compared with control mice, although the size of the tumors was not different. The effect of MMP-9 on lung tumor colonization was dependent on the expression of MMP-9 from bone marrow-derived cells and is most likely contributed by neutrophils. To examine temporal effects of stromal MMP-9, bioluminescence imaging from luciferase-expressing human lung cancer-derived A549 cells revealed that there were fewer tumor cells in the lungs of MMP-9 null mice as early as 19 hours after injection compared with control mice, with no difference in subsequent growth rates. Six hours after injection of tumor cells, MMP-9 null mice showed a 4-fold increase in the percent of tumor cells undergoing apoptosis compared with control mice. We conclude that MMP-9 from the bone marrow contributes to the early survival and establishment of tumors in the lung and has no effect on subsequent growth. These results provide insights into the failure of MMP inhibitors in clinical trials in patients with late-stage lung cancer.

Animals↗

PubMed2006

Matrix metalloproteinases: roles in cancer and metastasis.

The matrix metalloproteinase (MMP) family of extracellular proteinases play roles in normal physiological processes as well as in multiple disease settings including cancer. The link between MMP activity and cancer was considered strong enough to warrant considerable investment in pharmacological inhibitors of MMPs as a potential therapeutic modality, however, multiple large-scale clinical trials have all failed to reach their primary endpoints. This has led us to re-evaluate our thinking with respect to MMPs in cancer and shown that, most importantly, we need to understand the range of functions of these enzymes before we can effectively modulate them. The MMPs contribute to every stage of tumor progression, not just the later stages as was originally assumed. Additionally, through processing of their various substrates, MMP activity can have both pro- and anti-tumorigenic functions, thus their broad inhibition is likely to have unwanted consequences in some settings. Interactions between MMPs and proteinases of other classes are another important aspect of tumor biology and understanding these interactions is also necessary for development of effective therapeutic strategies. The aim of this article is to summarize recent findings in these areas and put them in the context of our growing understanding of how MMPs function in cancer development and progression.

Animals↗

PubMed2005

Increased plasma MMP9 in integrin alpha1-null mice enhances lung metastasis of colon carcinoma cells.

Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin alpha1-null mice, a genetic model for increased plasma levels of MMP9 and MMP9-generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202-7). We show that while the number of lung colonies in integrin alpha1-null mice was significantly increased compared to their wild-type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well-vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials.

Angiogenesis Inhibitors↗

PubMed2005

An orthotopic model of lung cancer to analyze primary and metastatic NSCLC growth in integrin alpha1-null mice.

The role of matrix metalloproteinase (MMP)9 in lung cancer progression is controversial. MMP9 promotes local tumor progression and distant metastasis in mouse models by enhancing extracellular matrix degradation, releasing VEGF from extracellular matrix and promoting vascular pericyte recruitment. Furthermore, increased plasma MMP9 expression levels in human subjects with metastatic non-small cell lung cancer (NSCLC) inversely correlates with survival. In contrast, MMP9 can benefit the host by generating inhibitors of endothelial cell proliferation such as angiostatin and NC1 domains of collagen IV. To better understand the role of host MMP9 on the primary growth and metastatic potential of NSCLC, we performed an orthotopic model of NSLC in integrin alpha1-null mice (a genetic model for increased MMP9). In these mice we observed decreased number, size and vascularization of primary NSCLC tumors when compared to wild type controls. In addition, decreased number and size of NSCLC-derived metastases were evident in the alpha1-null mice. Furthermore, pharmacological inhibition of MMPs in the alpha1-null mice at the time of tumor cell injection resulted in an increase in the number of both primary and metastatic lung cancer as compared to untreated mice, suggesting that primary growth and metastases of NSCLC are worsened by the early inhibition of MMPs. In conclusion, although MMP9 may potentially promote tumor growth and metastasis, production of MMP-dependent anti-angiogenic factors seems to override these effects and protects the host from NSCL growth and progression.

Animals↗

PubMed2005

MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL.

We developed a rodent model that mimics the osteoblastic and osteolytic changes associated with human metastatic prostate cancer. Microarray analysis identified MMP-7, cathepsin-K, and apolipoprotein D as being upregulated at the tumor-bone interface. MMP-7, which was produced by osteoclasts at the tumor-bone interface, was capable of processing RANKL to a soluble form that promoted osteoclast activation. MMP-7-deficient mice demonstrated reduced prostate tumor-induced osteolysis and RANKL processing. This study suggests that inhibition of MMP-7 will have therapeutic benefit in the treatment of prostate cancer-induced osteolysis.

Acid Phosphatase↗

PubMed2004

Proteinase activity in human and murine saliva as a biomarker for proteinase inhibitor efficacy.

As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.

Amidines↗

PubMed2004

Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells.

Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment as compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7(HIGH)-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium.

Animals↗

PubMed2004

Abnormal TNF activity in Timp3-/- mice leads to chronic hepatic inflammation and failure of liver regeneration.

Tumor-necrosis factor (TNF), a pleiotropic cytokine, triggers physiological and pathological responses in several organs. Here we show that deletion of the mouse gene Timp3 resulted in an increase in TNF-alpha converting enzyme activity, constitutive release of TNF and activation of TNF signaling in the liver. The increase in TNF in Timp3(-/-) mice culminated in hepatic lymphocyte infiltration and necrosis, features that are also seen in chronic active hepatitis in humans. This pathology was prevented when deletion of Timp3 was combined with Tnfrsf1a deficiency. In a liver regeneration model that requires TNF signaling, Timp3(-/-) mice succumbed to liver failure. Hepatocytes from Timp3(-/-) mice completed the cell cycle but then underwent cell death owing to sustained activation of TNF. This hepatocyte cell death was completely rescued by a neutralizing antibody to TNF. Dysregulation of TNF occurred specifically in Timp3(-/-), and not Timp1(-/-) mice. These data indicate that TIMP3 is a crucial innate negative regulator of TNF in both tissue homeostasis and tissue response to injury.

ADAM Proteins↗

PubMed2004

Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7-positive tumour by approx. 60%. Thus PB-M7VIS functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.

Amino Acid Sequence↗

PubMed2004

Tcf binding sequence and position determines beta-catenin and Lef-1 responsiveness of MMP-7 promoters.

The matrix metalloproteinase-7 (MMP-7) gene is a target of beta-catenin transactivation. Expression of the T-cell factor, Lef-1, enhances transcriptional activation of the human MMP-7 promoter by beta-catenin, but represses activation of the mouse MMP-7 promoter, both activities through consensus Tcf binding sites. The mouse promoter has a single Tcf binding element (mTBE) located downstream of the transcriptional start site, while the human promoter has two Tcf binding elements (hTBE1, hTBE2), both located upstream of the transcriptional start. hTBE1 and hTBE2 also differ in sequence from mTBE. Here we demonstrate that positioning of mTBE, upstream or downstream of the transcriptional start site dictated whether Lef-1 functioned as an activator or repressor, respectively. Sequence differences between mTBE and hTBE sites determined the potency of these activities, with hTBE sites being weaker. Mutational analysis of mTBE showed that increased Lef-1 activity mapped to G . C base pairings at 5' and 3' ends, and correlated with a threefold increase in Lef-1 binding affinity in vitro. Heterologous promoters with high affinity binding sites were 115-fold more responsive to beta-catenin than those with low affinity sites. Converting low affinity Tcf binding sites to high affinity sites increased beta-catenin responsiveness of the mouse and human promoters by 2-3 fold, and ectopic expression of Lef-1 increased beta-catenin responsiveness for promoters with low affinity binding sequences. We concluded that sequence and position of Tcf binding sites can determine the extent of beta-catenin-Lef-1 responsiveness for beta-catenin target genes.

Animals↗

PubMed2004

Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis.

We demonstrate a novel tumor-promoting role of myeloid immune suppressor Gr+CD11b+ cells, which are evident in cancer patients and tumor-bearing animals. These cells constitute approximately 5% of total cells in tumors. Tumors coinjected with Gr+CD11b+ cells exhibited increased vascular density, vascular maturation, and decreased necrosis. These immune cells produce high levels of MMP9. Deletion of MMP9 in these cells completely abolishes their tumor-promoting ability. Gr+CD11b+ cells were also found to directly incorporate into tumor endothelium. Consistent with this observation, Gr+CD11b+ cells acquire endothelial cell (EC) properties in tumor microenvironment and proangiogenic culture conditions. Our data provide evidence that Gr+CD11b+ cells of immune origin induced by tumors directly contribute to tumor growth and vascularization by producing MMP9 and differentiating into ECs.

Animals↗

PubMed2004

The influence of matrix metalloproteinase-7 on early mammary tumorigenesis in the multiple intestinal neoplasia mouse.

The multiple intestinal neoplasia (Min/+) mouse, which carries a mutant adenomatous polyposis coli (Apc) allele, is a model for human familial colon cancer. Like the human syndrome caused by mutant APC, the Min/+ mouse syndrome shows susceptibility to tumors of other tissues, including the mammary gland. The matrix metalloproteinase (MMP) MMP-7 (matrilysin) gene is transcriptionally induced by signal transduction pathways resulting from loss of APC function, and contributes to the progression of benign and malignant intestinal epithelial cells. Mammary tumors that develop in Min/+ mice express MMP-7. To investigate whether mutant APC and MMP-7 can cooperate in mammary tumorigenesis, we compared N-ethyl-N-nitrosourea (ENU)-enhanced mammary tumor formation in Min/+ mice that were either wild-type or deficient in MMP-7. Min/+ mice lacking MMP-7 demonstrate a 60% reduction in the number of early focal lesions in the mammary gland at early, but not later, timepoints. We conclude that MMP-7 transiently influences early stage mammary tumorigenesis.

Animals↗

PubMed2003

Matrix metalloproteinase inhibitors for cancer therapy:the current situation and future prospects.

Inhibition of matrix metalloproteinases (MMPs), a family of proteolytic enzymes linked to many aspects of cancer progression, has been explored as a therapeutic goal for almost two decades. Thus far, all tested MMP inhibitors (MMPIs) have failed to reach primary end points in Phase III clinical trials, although secondary analyses suggest benefits in particular patient groups. The clinical development of these agents has been hampered by problems related to determination of effective dosages and side effects that necessitate dose lowering or drug holidays. Imaging technologies offer hope as a means to measure enzyme activity and hence effective enzyme inhibition in vivo. Meanwhile, recent results from genetic studies of both mice and man have given some clues to possible causes of musculoskeletal side effects. Future progress in the therapeutic use of MMPIs is dependent on the ability to selectively target cancer-associated MMPs at the correct stage in tumour progression and the development of surrogate markers of in vivo efficacy.

Antineoplastic Agents↗

PubMed2003

CMT-3. CollaGenex.

CMT-3 is an orally active matrix metalloprotease inhibitor, and one of a series of inhibitors of multiple proteases and cytokines, under development by CollaGenex. The compound is currently undergoing phase II trials for the potential treatment of cancer, in particular metastatic cancer and HIV-related Kaposi's sarcoma.

Animals↗

PubMed2002

Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand.

Soluble Fas ligand (sFasL) is released from the cell surface by matrix metalloproteinases (MMPs), one of which is MMP-7. We have reported that MMP-7-generated sFasL is pro-apoptotic in both in vitro and in vivo systems. However, there are contradictory reports that the soluble form of FasL is inactive or anti-apoptotic, resulting in significant controversy in the literature. One potential explanation for these discrepancies is that forms of sFasL with different amino-terminal sequences have been demonstrated to have varying activities. Here we report that MMP-7 cleaves murine and human FasL at sites that are distinct from previously reported cleavage sites resulting in production of novel forms of sFasL. Cleavage of FasL by MMP-7 occurs at the leucine residues in the sequence "ELAELR" within the region between the transmembrane and trimerization domains. When this site is unavailable, a more c-terminal site, "SL," is cleaved. MMP-7 differentially processes murine and human FasL since it cleaves human FasL not only at the "ELAELR" site but also at a previously identified site. Additionally, MMP-3, but not MMP-2, was found to have the same cleavage specificity for murine FasL as MMP-7. We conclude that the controversy regarding the biological activity of sFasL may be explained, in part, by the generation of distinct forms of sFasL as a result of cleavage at specific sequences.

Amino Acid Sequence↗

PubMed2002

Matrilysin (matrix metalloproteinase-7) selects for apoptosis-resistant mammary cells in vivo.

Overexpression of the matrix metalloproteinase matrilysin (matrix metalloproteinase-7) in the mouse mammary gland promotes mammary hyperplasia and accelerates the onset of oncogene-induced mammary tumors. In cell culture models, acute exposure of cells coexpressing Fas and Fas ligand (FasL) to matrilysin induces apoptosis, whereas chronic exposure to matrilysin selects for apoptosis-resistant cells. We now demonstrate that matrilysin promotes resistance to apoptosis in vivo. Matrilysin expression increased apoptosis in the involuting mammary gland of mice that had undergone a single pregnancy and lactation cycle. Premature basement membrane disruption was detected in matrilysin-expressing mice, which could account for the increase in apoptosis. However, multiparous mice, in which the involuting mammary epithelial cells have been repeatedly exposed to matrilysin, show a significant decrease in apoptosis. Mammary tissue from multiparous matrilysin-expressing mice showed decreased FasL expression, suggesting that loss of FasL is at least one mechanism of matrilysin-induced resistance to apoptosis. We propose that matrilysin promotes mammary tumor formation by enhancing the selection of cells that are resistant to apoptosis.

Animals↗

PubMed2002

Matrix metalloproteinase inhibitors and cancer: trials and tribulations.

For at least 30 years, matrix metalloproteinases (MMPs) have been heralded as promising targets for cancer therapy on the basis of their massive up-regulation in malignant tissues and their unique ability to degrade all components of the extracellular matrix. Preclinical studies testing the efficacy of MMP suppression in tumor models were so compelling that synthetic metalloproteinase inhibitors (MPIs) were rapidly developed and routed into human clinical trials. The results of these trials have been disappointing. Here we review the studies that brought MPIs into clinical testing and discuss the design and outcome of the trials in light of new information about the cellular source, substrates, and mode of action of MMPs at different stages of tumor progression. The important lessons learned from the MPI experience may be of great value for future studies of MPIs and for cancer drug development in general.

Animals↗