Epithelial nuclear factor-κB signaling promotes lung carcinogenesis via recruitment of regulatory T lymphocytes.

The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase ß in airway epithelium (IKTA (IKKß trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.

Matrix metalloproteinases as targets for therapy in Kaposi sarcoma.

Acquired immune deficiency syndrome-associated Kaposi sarcoma is a progressive and occasionally fatal condition. The strong angiogenic component of this disease makes it particularly suitable for treatment with the emerging class of drugs that act as antiangiogenic agents. Matrix metalloproteinases have been shown to play prominent roles in the angiogenic process, and small molecule inhibitors of these enzymes are currently being tested as antiangiogenic agents in various malignancies. Given that matrix metalloproteinases contribute to multiple steps of the angiogenic process, inhibitors of these enzymes, either alone or in combination with other agents, may represent a particularly effective therapeutic approach for Kaposi sarcoma.

The PEA3 subfamily of Ets transcription factors synergizes with beta-catenin-LEF-1 to activate matrilysin transcription in intestinal tumors.

The matrix metalloproteinase matrilysin (MMP-7) is expressed in the tumor cells of a majority of mouse intestinal and human colonic adenomas. We showed previously that matrilysin is a target gene of beta-catenin-Tcf, the transcription factor complex whose activity is thought to play a crucial role in the initiation of intestinal tumorigenesis. Here we report that overexpression of a stable mutant form of beta-catenin alone was not sufficient to effect expression of luciferase from a matrilysin promoter-luciferase reporter plasmid. However, cotransfection of the reporter with an expression vector encoding the PEA3 Ets transcription factor, or its close relatives ER81 and ERM, increased luciferase expression and rendered the promoter responsive to beta-catenin-LEF-1 as well as to the AP-1 protein c-Jun. Other Ets proteins could not substitute for the PEA3 subfamily. Luciferase activity was induced up to 250-fold when PEA3, c-Jun, beta-catenin, and LEF-1 were coexpressed. This combination of transcription factors was also sufficient to induce expression of the endogenous matrilysin gene. Furthermore, all matrilysin-expressing benign intestinal tumors of the Min mouse expressed a member of the PEA3 subfamily, as did all human colon tumor cell lines examined. These data suggest that the expression of members of the PEA3 subfamily, in conjunction with the accumulation of beta-catenin in these tumors, leads to coordinate upregulation of matrilysin gene transcription, contributing to gastrointestinal tumorigenesis.

Matrilysin [MMP-7] expression selects for cells with reduced sensitivity to apoptosis.

The matrix metalloproteinase matrilysin (MMP-7) has been demonstrated to contribute to tumor development. We have shown previously that members of the TNF family of apoptosis-inducing proteins are substrates for this enzyme, resulting in increased death pathway signaling. The goal of the current study was to reconcile the proapoptotic and tumor-promoting functions of matrilysin. In the human HBL100 and murine NMuMG cell lines that represent early stages of tumor progression and that express both Fas ligand and its receptor, exposure to matrilysin results in cell death that can be blocked by FasL neutralizing antibodies. Constitutive expression of matrilysin in these cell lines selects for cells with reduced sensitivity to Fas-mediated apoptosis as demonstrated both with a receptor-activating antibody and with in vitro activated splenocytes. Matrilysin-expressing cells are also significantly less sensitive to chemical inducers of apoptosis. We propose that the expression of matrilysin that has been reported at early stages in various tumor types can act to select cells with a significantly decreased chance of removal due to immune surveillance. As a result, these cells are more likely to acquire additional genetic modifications and develop further as tumors.

Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.

The function of many transmembrane molecules can be altered by cleavage and subsequent release of their ectodomains. We have investigated ectodomain cleavage of the cell-cell adhesion and signal-transducing molecule E-cadherin. The E-cadherin ectodomain is constitutively shed from the surface of MCF-7 and MDCKts.srcC12 cells in culture. Release of the 80 kDa soluble E-cadherin fragment is stimulated by phorbol-12-myristate-13-acetate and is inhibited by overexpression of the tissue inhibitor of metalloproteinases-2. The metalloproteinases matrilysin and stromelysin-1 both cleave E-cadherin at the cell surface and release sE-CAD into the medium. The soluble E-cadherin fragment thus released inhibits E-cadherin functions in a paracrine way, as indicated by induction of invasion into collagen type I and inhibition of E-cadherin-dependent cell aggregation. Our results, therefore, suggest a novel mechanism by which metalloproteinases can influence invasion.

Matrix metalloproteinases: biologic activity and clinical implications.

Tumor progression is a complex, multistage process by which a normal cell undergoes genetic changes that result in phenotypic alterations and the acquisition of the ability to spread and colonize distant sites in the body. Although many factors regulate malignant tumor growth and spread, interactions between a tumor and its surrounding microenvironment result in the production of important protein products that are crucial to each step of tumor progression. The matrix metalloproteinases (MMPs) are a family of degradative enzymes with clear links to malignancy. These enzymes are associated with tumor cell invasion of the basement membrane and stroma, blood vessel penetration, and metastasis. They have more recently been implicated in primary and metastatic tumor growth and angiogenesis, and they may even have a role in tumor promotion. This review outlines our current understanding of the MMP family, including the association of particular MMPs with malignant phenotypes and the role of MMPs in specific steps of the metastatic cascade. As scientific understanding of the MMPs has advanced, therapeutic strategies that capitalize on blocking the enzymes have rapidly developed. The preclinical and clinical evolution of the synthetic MMP inhibitors (MMPIs) is also examined, with the discussion encompassing important methodologic issues associated with determining clinical efficacy of MMPIs and other novel therapeutic agents.

Matrix metalloproteinase-3-dependent generation of a macrophage chemoattractant in a model of herniated disc resorption.

Herniated disc (HD) is a common health problem that is resolved by surgery unless spontaneous resorption occurs. HD tissue contains abundant macrophage infiltration and high levels of matrix metalloproteinases (MMPs) MMP-3 and MMP-7. We developed a model system in which disc tissue or isolated chondrocytes from wild-type or MMP-null mice were cocultured with peritoneal macrophages and used this system to investigate the role of MMPs and chondrocyte/macrophage interactions in disc resorption. We observed a marked enhancement of MMP-3 protein and mRNA in chondrocytes after exposure to macrophages. Chondrocytic MMP-3, but not MMP-7, was required for disc resorption, as determined by assaying for a reduction in wet weight and proteoglycan content after 3 days of coculture. Surprisingly, chondrocyte MMP-3 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by proteolytically active macrophages. We conclude that macrophage induction of chondrocyte MMP-3 plays a major role in disc resorption by mechanisms that include the generation of a bioactive macrophage chemoattractant.

Matrix metalloproteinase-7-dependent release of tumor necrosis factor-alpha in a model of herniated disc resorption.

Herniated disc (HD), one of the major causes of low back pain, is often resolved spontaneously without surgical intervention. Resorption is associated with a marked increase in infiltrating macrophages, and the matrix metalloproteinases (MMP) MMP-3 and MMP-7 have been implicated in this phenomenon. We developed a murine organ culture model in which intact intervertebral discs were cocultured with peritoneal macrophages to investigate the role of MMPs in HD resorption. Using macrophages isolated from MMP-null mice, we report that macrophage-produced MMP-7 was required for proteoglycan degradation, loss of wet weight, and macrophage infiltration of cocultured discs. The inability of MMP-7-deficient macrophages to infiltrate discs could not be attributed to a defect in macrophage migration. MMP-7 was required for the release of the cytokine TNF-alpha from peritoneal macrophages. The generation of soluble TNF-alpha was essential for the induction of MMP-3 in disc cocultures, which in turn is required for the generation of a macrophage chemoattractant and subsequent macrophage infiltration. TNF-alpha release from macrophages was necessary but insufficient for disc resorption, which required macrophage infiltration. We conclude that there is extensive communication between macrophages and chondrocytes in HD resorption and that an essential component of this communication is the requirement for MMPs to release soluble bioactive factors.

The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis.

BACKGROUND: The Fas ligand/Fas receptor (FasL/Fas) system is an important mediator of apoptosis in the immune system where the juxtaposition of cells expressing the cell-surface ligand induces the apoptotic pathway in Fas-expressing lymphocytes. The FasL/Fas system has also been shown to be involved in apoptosis in epithelial tissues, including the involuting rodent prostate. FasL can be shed through the action of an hitherto unidentified metalloproteinase to yield soluble FasL (sFasL), although the biological activity of sFasL has been disputed. RESULTS: Here we report that the matrix metalloproteinase matrilysin can process recombinant and cell-associated FasL to sFasL, and that matrilysin-generated sFasL was effective at inducing apoptosis in a target epithelial cell population. In the involuting mouse prostate, FasL and matrilysin colocalized to the cell surface in a restricted population of epithelial cells. Mice deficient in matrilysin demonstrated a 67% reduction in the apoptotic index in the involuting prostate compared with wild-type animals, implicating matrilysin in this FasL-mediated process. CONCLUSIONS: The results show that a functional form of sFasL was generated by the action of the metalloproteinase matrilysin, and suggest that matrilysin cleavage of FasL is an important mediator of epithelial cell apoptosis.

The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors.

Matrilysin is a matrix metalloproteinase expressed in the tumor cells of greater than 80% of intestinal adenomas. The majority of these intestinal tumors are associated with the accumulation of beta-catenin, a component of the cadherin adhesion complex and, through its association with the T Cell Factor (Tcf) DNA binding proteins, a regulator in the Wnt signal transduction pathway. In murine intestinal tumors, matrilysin transcripts show striking overlap with the accumulation of beta-catenin protein. The matrilysin promoter is upregulated as much as 12-fold by beta-catenin in colon tumor cell lines in a manner inversely proportional to the endogenous levels of beta-catenin/Tcf complex and is dependent upon a single optimal Tcf-4 recognition site. Coexpression of the E-cadherin cytoplasmic domain blocked this induction and reduced basal promoter activity in every colon cancer cell line tested. Inactivation of the Tcf binding site increased promoter activity and overexpression of the Tcf factor, LEF-1, significantly downregulated matrilysin promoter activity, suggesting that beta-catenin transactivates the matrilysin promoter by virtue of its ability to abrogate Tcf-mediated repression. Because genetic ablation of matrilysin decreases tumor formation in multiple intestinal neoplasia (Min) mice, we propose that regulation of matrilysin production by beta-catenin accumulation is a contributing factor to intestinal tumorigenesis.

Matrilysin in early stage intestinal tumorigenesis.

The demonstration that matrix metalloproteinases [MMPs] play an active role in the invasion and metastasis stages of tumor progression has led to the development of a new class of anti-metastatic chemotherapeutic agent, the matrix metalloproteinase inhibitors [MMPIs]. We present evidence to suggest that the MMP matrilysin, in particular, plays an essential role in much earlier stages of intestinal tumorigenesis. Matrilysin is detected in a high percentage of pre-invasive lesions, in contrast to its absence in most normal tissues, and is expressed by the epithelial-derived tumor cells. Manipulating levels of this enzyme in vitro results in cell lines with enhanced tumorigenic potential, while ablating the gene in vivo leads to a significant reduction in tumor number in two different animal models of intestinal tumorigenesis. Additionally, regulation of matrilysin gene expression appears to be under the control of genetic pathways which are activated very early in the tumor development sequence. Although the precise mechanism by which matrilysin activity contributes to tumor formation is not yet clear, we propose that MMPIs may be of benefit as chemopreventative agents in addition to their therapeutic potential for metastatic disease.

Role of matrix metalloproteinases in invasion and metastasis: biology, diagnosis and inhibitors.

The processes of tumour invasion and subsequent metastasis are the most lethal aspects of cancer. Whilst many factors are involved, the matrix metalloproteinases (MMPs) have been implicated as key-rate limiting enzymes in the invasive process. This family consisting of eight members of similar structure, can be roughly divided into three groups based on substrate specificity. All are secreted in a latent form and require proteolytic cleavage for activation. The expression of these enzymes is regulated at the transcriptional level by a variety of growth factors and oncogenes. They are also regulated at the protein level by a family of specific inhibitors called the tissue inhibitors of metalloproteinases (TIMPs). Studies in human tumour samples have shown a positive correlation between metalloproteinase expression and metastatic potential. The levels of metalloproteinase expression have been manipulated using molecular biology techniques in several cell lines and shown a similar correlation. These results suggest that an understanding of metalloproteinase expression and proteolytic activity may lead to the development of effective therapeutic agents with the potential to reduce the incidence of metastatic cancer.