Matrix metalloproteinases (MMP) have been implicated in virtually all aspects of tumor progression. However, the recent failure of clinical trials employing synthetic MMP inhibitors in cancer chemotherapy has led us to hypothesize that some MMPs may actually serve the host in its defense against tumor progression. Here we show that mice deficient in macrophage elastase (MMP-12) develop significantly more gross Lewis lung carcinoma pulmonary metastases than their wild-type counterparts both in spontaneous and experimental metastasis models. The numbers of micrometastases between the two groups are equivalent; thus, it seems that MMP-12 affects lung tumor growth, and not metastasis formation, per se. MMP-12 is solely macrophage derived in this model, being expressed by tumor-associated macrophages and not by tumor or stromal cells. The presence of MMP-12 is associated with decreased tumor-associated microvessel density in vivo and generates an angiostatic>angiogenic tumor microenvironment that retards lung tumor growth independent of the production of angiostatin. These data define a role for MMP-12 in suppressing the growth of lung metastases and suggest that inhibitors designed to specifically target tumor-promoting MMPs may yet prove effective as cancer therapeutics. (Cancer Res 2006; 66(12): 6149-55)
Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.
Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor-positive (ER) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in -mutant ER breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in mutant ER breast cancer. Potential eligible patients with clinical stage II/III ER/HER2 breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor sequencing. Patients positive for mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17. Fifty-one patients preregistered and 16 of 22 with -mutant tumors received study drug. Three patients went off study due to C1D17 Ki67>10% ( = 2) and toxicity ( = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an mutation at surgery. MK-2206 is unlikely to add to the efficacy of anastrozole alone in -mutant ER breast cancer and should not be studied further in the target patient population. .
The serine proteinases plasmin and thrombin convert proenzyme matrix metalloproteinases (MMPs) into catalytically active forms. In addition, we demonstrate that plasmin(ogen) and thrombin induce a significant increase in secretion of activated murine macrophage elastase ( Serine and matrix metalloproteinases (MMPs)1 have important roles in hemostasis and remodeling of extracellular matrices during fibrinolysis and tissue repair. Abnormal regulation of these proteinases may cause tissue destruction. MMPs are typically secreted in zymogen form and require extracellular activation. The serine proteinases plasminogen, plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA) have classically been shown to function in fibrinolysis (tPA/uPA/plasmin), cell motility and invasion (uPA/plasmin), and MMP activation. The MMPs are a family of structurally related zinc-containing enzymes that are either secreted or membrane-associated (membrane-type MMPs; MT-1-5 MMPs). As a group, the MMPs are capable of degrading all extracellular matrix components.In addition to fibrinolysis and extracellular matrix degradation, MMPs and serine proteinases can cleave nonmatrix proteins with significant biological ramifications. For example, MMPs and related ADAMs (a disintegrin and metalloproteinase domain) can cleave and release a variety of active molecules from cell surfaces, such as tumor necrosis factor-␣ (1), and plasmin can activate transforming growth factor- in plasma (2). In addition, both MMPs and serine proteinases generate angiostatin, an inhibitor of endothelial cell proliferation, from plasminogen (3-9).Such redundant activity and interaction of the serine and metalloproteinase families have precedence in previous work. Plasmin cleaves many pro-MMPs, including the gelatinases MMP-2 and -9 (progelatinase A and B) (10) and MMP-12 (macrophage elastase) (11) within the N-terminal domain, altering conformation and exposing the active site zinc, which in turn releases the remainder of the proenzyme domain, resulting in a fully active MMP. At the cellular level, uPA-generated plasmin controls gelatinase activity in HT1080 cells (10). Carmeliet et al. (11) have recently shown that MMP activation in macrophages in culture is uPA/plasmin-dependent, and data suggests similar activation in a murine model of atherosclerotic microaneurysm formation. Another serine proteinase, thrombin, has also been reported to activate MMPs (progelatinase A, MMP-2) in microvascular endothelial cells (12) and to actually increase collagenase (MMP-1) protein and stromelysin (MMP-3) mRNA and protein in large vessel endothelial cells (human saphenous vein and mammary artery) (13).Protease-activated receptor (PAR-1) is a unique cell surfaceassociated receptor activated by thrombin proteolysis that is characteristically coupled to G protein heterodimers (14) and is also activated by plasmin (15). It is a member of the seventransmembrane domain family that is activated following proteolytic cleavage of its N terminus (16...
Elastic fibers are composed of the protein elastin and a network of 10 -12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1. MAGP-2 was found complexed with the Jagged1 extracellular domain shed from 293T cells and COS-7 cells coexpressing fulllength Jagged1 and MAGP-2. MAGP-2 shedding of the Jagged1 extracellular domain was decreased by the metalloproteinase hydroxamate inhibitor BB3103 implicating proteolysis in its release. Although MAGP-2 also interacted with the other DSL ligands, Jagged2 and Delta1, they were not found associated with MAGP-2 in the conditioned media, identifying differential effects of MAGP-2 on DSL ligand shedding. The related microfibrillar protein MAGP-1 was also found to interact with DSL ligands but, unlike MAGP-2, was unable to facilitate the shedding of Jagged1. Our findings suggest that in addition to its role in microfibrils, MAGP-2 may also affect cellular differentiation through modulating the Notch signaling pathway either by binding to cell surface DSL ligands or by facilitating release and/or stabilization of a soluble extracellular form of Jagged1.
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