Intradomain Cleavage of Inhibitory Prodomain Is Essential to Protumorigenic Function of Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) in Vivo

Abstract

Invasive cancers use pericellular proteolysis to breach the extracellular matrix and basement membrane barriers and invade the surrounding tissue. Proinvasive membrane type-1 matrix metalloproteinase (MT1-MMP) is the primary mediator of proteolytic events on the cancer cell surface. MT1-MMP is synthesized as a zymogen. The latency of MT1-MMP is maintained by its N-terminal inhibitory prodomain. In the course of MT1-MMP activation, the R 108 RKR 111 2Y 112 prodomain sequence is processed by furin. The intact prodomain released by furin alone, however, is a potent inhibitor of the emerging MT1-MMP enzyme. Evidence suggests that the prodomain undergoes intradomain cleavage at the PGD2L 50 site followed by the release of the degraded prodomain by furin cleavage that finalizes the two-step activation event. Normal and malignant cells rely on pericellular proteolysis to penetrate through tissue barriers (1-3). The invasive phenotype is frequently associated with the cellular matrix metalloproteinase (MMP) 2 activity (4). The human MMP family comprises 24 individual enzymes (5). Six membrane type MMPs (MT-MMPs) are distinguished from soluble MMPs by an additional transmembrane domain and a short cytoplasmic tail (MT1-, MT2-, MT3-, and MT5-MMP) or by a glycosylphosphatidylinositol anchor (MT4-and MT6-MMPs) (6, 7). There is compelling evidence that MT1-MMP functions as a proinvasive factor (8 -10). MT1-MMP is the primary mediator of proteolytic events on the cell surface (11). MT1-MMP is directly involved in the pericellular proteolysis of the ECM proteins, activation of soluble MMPs, and cleavage of adhesion and signaling cell receptors (12-14). Although MT1-MMP is ubiquitously expressed, its activity is elevated in metastatic malignancies (15), a cause of mortality and morbidity in cancer patients.MT1-MMP is synthesized de novo as a latent proenzyme (16). Proteolytic removal of the N-terminal prodomain is required for the proenzyme conversion into the functionally active MT1-MMP enzyme. It is established that in the course of the secretion pathway of the MT1-MMP proenzyme through the cell compartment the R 108 RKR 111 2Y 112 prodomain sequence is processed by furin (17,18). We demonstrated, however, that the intact prodomain released by furin alone is a potent inhibitor of the emerging MT1-MMP enzyme. Evidence suggests that the activation of cellular MT1-MMP represents a stepwise process (19,20). In this stepwise process, the PGD2L 50 sequence of the prodomain "bait region" is cleaved intracellularly either by MMPs, which are distinct from MT1-MMP, or by autocatalysis if cellular MT1-MMP is overexpressed. Intradomain cleavage results in the activation intermediate commencing from the N-terminal Leu 50 . Furin proteolysis then follows to complete both the degradation and removal of the residual prodomain sequence, thus transforming the activation intermediate into the mature, proteolytically active MT1-MMP enzyme commencing from the N-terminal Tyr 112 (13,21,22). We used mutagenesis to inactivate the PGD2L 50 cleavage s...

Novel MT1-MMP Small-Molecule Inhibitors Based on Insights into Hemopexin Domain Function in Tumor Growth

“…184B5 cells stably transfected with the original pLenti6/V5-D-TOPO lentiviral vector (184B5 cells) or the lentiviral vector encoding the MT1-MMP C-terminally tagged with a V5 tag (185B5-MT cells) were constructed earlier (30). MCF-7 cells stably expressing the wild type MT1-MMP (MCF7-MT cells), MT1-MMP C-terminally tagged with a V5 tag (MCF7-MT-V5 cells) or the β 3 integrin subunit (MCF7-β3/zeo cells) alone or co-expressing the latter with MT1-MMP (MCF7-β3/MT cells) or the MT1-MMP mutant lacking the 319–508 PEX sequence (MCF7-β3/ΔPEX cells) were obtained earlier (12, 27, 30–32). …”

“…The integrity of the RNA samples was validated using an Experion automated electrophoresis system (Bio-Rad). The RNA templates (50 ng) were used in the 25-μl RT-PCR reactions supplemented with the corresponding forward and reverse primers (0.6 μM each) using the OneStep RT-PCR system ( Qiagen ) (12). After the completion of the first strand synthesis, RT-PCR was performed for 35 cycles (denaturation at 95°C, annealing at 55°C and elongation at 72°C; 30, 30 and 60 sec, respectively).…”

“…The MT1-MMP multi-domain structure includes the N-terminal prodomain, the catalytic domain (CAT), the hinge linker, the hemopexin domain (PEX), the transmembrane and the C-terminal cytoplasmic domains (11). MT1-MMP is synthesized as a latent zymogen that requires proteolytic processing of the N-terminal inhibitory prodomain to generate the mature enzyme (12, 13). …”

Proteolysis during Tumor Cell Extravasation In Vitro: Metalloproteinase Involvement across Tumor Cell Types

“…It is thought that MT1-MMP is activated by the proprotein convertase furin, although furin-independent activation of MT1-MMP has been reported [8–10]. Golubkov et al demonstrated the importance of the furin-mediated activation of MT1-MMP for tumorigenicity [11], while others used a small molecule inhibitor of the process to reduce the invasiveness of HT1080 cells [12]. Active furin cycles between the Golgi and the cell surface leading to MT1-MMP activation at both locations [9,13,14].…”

Non-destructive and Selective Imaging of the Functionally Active, Pro-invasive Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Enzyme in Cancer Cells

“…There is a consensus among researchers that pro-invasive MT1-MMP is a key element in tumor cell migration and pericellular proteolysis (7)(8)(9)(10)(11). To become catalytically active, the latent MT1-MMP proenzyme requires proteolytic removal of its inhibitory prodomain (12)(13)(14)(15)(16). The active MT1-MMP enzyme cleaves extracellular matrix components, cell adhesion, and signaling receptors, and also initiates the activation pathway of soluble MMP-2 and MMP-13 (17)(18)(19)(20)(21)(22)(23)(24).…”