Data from Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer
Anika AgarwalLidija CovicLeila M. SevignyNicole C. KaneiderKatherine LazaridesGissou AzabdaftariSheida SharifiAthan Kuliopulos
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<div>Abstract<p>Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an <i>in vivo</i> proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer. [Mol Cancer Ther 2008;7(9):2746–57]</p></div>Keywords:
Tumor progression
Matrix metalloproteinase 9
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Matrix Metalloproteinase 3
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The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.
Matrix metalloproteinase inhibitor
Gelatinase A
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Matrix metalloproteinase inhibitor
Matrix metalloproteinase 9
Matrix (chemical analysis)
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The study of meningiomas by insitu hybridization and immunohistochemistry shows that matrix metalloproteinase-2,metalloproteinase-9 (MMP-2,MMP-9) have an abnormal expression in meningiomas tissues,which indicated that they are related to invasion of meningiomas.Tissue inhibitor of metalloproteinase (TIMP-1,TIMP-2) can inhibit the activity of MMPs,which indicated that the balance between MMPs and TIMPs is the important factor affecting the invasion of meningiomas.So further studies about the expression of MMPS and TIMPs in meningiomas and its regulating mechanism,as well as how to inhibit the invasion of meningiomas by inhibiting the activity of MMPs,which will be a new method in treating meningiomas.
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Matrix metalloproteinase inhibitor
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Previous studies have shown that membrane type 1-matrix metalloproteinase (MT1-MMP) (MMP-14) initiates pro-MMP-2 activation in a process that is tightly regulated by the level of tissue inhibitor of metalloproteinase (TIMP)-2. However, given the difficulty in modulating TIMP-2 levels, the direct effect of TIMP-2 on MT1-MMP processing and on pro-MMP-2 activation in a cellular system could not be established. Here, recombinant vaccinia viruses encoding full-length MT1-MMP or TIMP-2 were used to express MT1-MMP alone or in combination with various levels of TIMP-2 in mammalian cells. We show that TIMP-2 regulates the amount of active MT1-MMP (57 kDa) on the cell surface whereas in the absence of TIMP-2 MT1-MMP undergoes autocatalysis to a 44-kDa form, which displays a N terminus starting at Gly285 and hence lacks the entire catalytic domain. Neither pro-MT1-MMP (N terminus Ser24) nor the 44-kDa form bound TIMP-2. In contrast, active MT1-MMP (N terminus Tyr112) formed a complex with TIMP-2 suggesting that regulation of MT1-MMP processing is mediated by a complex of TIMP-2 with the active enzyme. Consistently, TIMP-2 enhanced the activation of pro-MMP-2 by MT1-MMP. Thus, under controlled conditions, TIMP-2 may act as a positive regulator of MT1-MMP activity by promoting the availability of active MT1-MMP on the cell surface and consequently, may support pericellular proteolysis.
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Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.
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Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, we replaced the C-terminal domain of TIMP-1 with those of TIMP-2, -3 and -4 to create a series of "T1:TX" chimeras. The affinity of the chimeras against ADAM10, ADAM17, MMP14 and MMP19 was investigated. We can show that replacement of the C-terminal domain by those of other TIMPs dramatically increased the affinity of TIMP-1 for some MPs. Furthermore, the chimeras were able to suppress TNF-α and HB-EGF shedding in cell-based setting. Unlike TIMP-1, T1:TX chimeras had no growth-promoting activity. Instead, the chimeras were able to inhibit cell migration and development in several cancer cell lines. Our findings have broadened the prospect of TIMPs as cancer therapeutics. The approach could form the basis of a new strategy for future TIMP engineering.
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Interstitial collagenase
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Although tissue inhibitor of metalloproteinase‐2 (TIMP‐2) is known to be not only an inhibitor of matrix metalloproteinases (MMP) but also a cofactor for membrane‐type 1 MMP (MT1‐MMP)‐mediated MMP‐2 activation, it is still unclear how TIMP‐2 regulates MMP‐2 activation and cleavage of substrates by MT1‐MMP. In the present study we examined the levels of cell‐surface MT1‐MMP, MMP‐2 activation and cleavage of MT1‐MMP substrates in 293T cells transfected with the MT1‐MMP and TIMP‐2 genes. Co‐expression of TIMP‐2 at an appropriate level increased the level of cell‐surface MT1‐MMP, both the TIMP‐2‐bound and free forms, and generated processed MMP‐2 with gelatin‐degrading activity. In contrast, MT1‐MMP substrates testican‐1 and syndecan‐1 were cleaved by the cells expressing MT1‐MMP, which was inhibited by TIMP‐2 even at levels that stimulate MMP‐2 activation. These results suggest that TIMP‐2 environment determines MT1‐MMP substrate choice between direct cleavage of its own substrates and MMP‐2 activation. ( Cancer Sci 2007; 98: 563–568)
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Matrix metalloproteinase inhibitor
Gelatinase A
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