Aberrant, persistent inclusion into lipid rafts limits the tumorigenic function of membrane type-1 matrix metalloproteinase in malignant cells
Introduction
The sustained presence of matrix metalloproteinases (MMPs) in a tumor environment produced both by the stromal cells and the cancer cells leads to destruction of the normal extracellular matrix (ECM) and the remodeling of tissue [1]. Matrix metalloproteinases (MMPs) are a comprehensive family of zinc-enzymes that degrade the ECM and the cell surface molecules[2], [3], [4]. Among known human MMPs, membrane-tethered MMPs have been shown to be the most important in promoting tumor cell migration and invasion [5], [6], [7].
Membrane-tethered MT1-MMP, the most abundant member of the membrane-type (MT) matrix metalloproteinase subfamily, is distinguished from soluble MMPs by a relatively short transmembrane domain and a cytoplasmic tail (CT) [8]. These domains associate the protease with discrete regions of the plasma membrane and the intracellular compartment. This protease functions in cancer cells as the main mediator of proteolytic events on the cell surface, and is directly involved in the cleavage of cell surface receptors and the focused pericellular proteolysis of the ECM components [9], [10], [11]. Thus, in addition to MT1-MMP's ability to degrade matrix components directly as well as to initiate the activation pathway of at least two soluble secretory MMPs, proMMP-2 and proMMP-13 (thereby further promoting degradation of the ECM) [12], [13], MT1-MMP is involved in the cleavage of cell surface receptors including CD44 [14], tissue transglutaminase [15], E-cadherin [16], [17], and the precursor of integrin αV subunit [18], [19]. This activity allows the cancer cells to adjust their receptor profile to the continually changing matrix environment. An understanding of the function and the regulation of MT1-MMP in carcinogenesis is absolutely necessary for the design of novel and successful, potent anticancer pharmaceuticals [9], [10], [11], [20].
The functional activity of MT1-MMP is controlled by furin-dependent processing [21], [22] and autolytic activation [23], [24], [25], [26], by inhibition by tissue inhibitors of matrix metalloproteinases [27], and by a self-proteolysis regulatory pathway [28]. Additionally, the cell-surface-associated activity of MT1-MMP is regulated by trafficking through the cell compartment to the plasma membrane and internalization of the protease into the transient endosome-like compartment inside the cells [29]. These mechanisms jointly govern the presentation of MT1-MMP at cell surfaces. Recently, the trafficking of MT1-MMP has emerged as a critical issue in its biological function [30], [31], [32]. Our earlier findings suggested that the CT peptide sequence is essential to the recruitment of MT1-MMP to the cell surface, apparently because this sequence is involved in the mechanisms of trafficking and internalization of the protease [33]. Understanding these mechanisms is essential for the design of improved, efficient anticancer therapies that involve the inhibitors of the MT1-MMP function. These conclusions prompted us to investigate in more detail the role of the CT in regulating the cell surface expression of MT1-MMP in cancer cells of distinct tissue origin such as breast carcinoma and glioma cells.
In this report, we showed that in contrast to the wild-type enzyme, mutant MT1-MMP lacking the C-terminal cytoplasmic tail was associated with caveolae-enriched lipid rafts in breast carcinoma cells. The recruitment of the MT1-MMP activity to caveolae-enriched lipid rafts strongly limited the tumorigenic function of the protease. Our observations lead us to hypothesize that a stringent regulation of trafficking and internalization is essential for the functional role of MT1-MMP in tumor cells, and that the CT plays a pivotal role in the implementation of these regulatory mechanisms. Our novel data support the hypothesis that MT1-MMP plays a critical role in tumor progression [34] and highlight the mechanisms that control the presentation, trafficking, and function of this membrane-tethered protease.
Section snippets
Antibodies and reagents
All reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated. Goat antirabbit IgG conjugated with horseradish peroxidase (HRP), rabbit antibody AB815 against the hinge region of MT1-MMP, the TMB/M substrate for HRP and GM6001 (a broad-range hydroxamate inhibitor of MMP activity) were from Chemicon International (Temecula, CA). Rabbit antibody against the recombinant catalytic domain of MT1-MMP was generated in our laboratory [33]. A rabbit antibody to caveolin-1 was from
The C-end truncated MT1-MMP enzyme is highly potent in self-proteolysis and in activating proMMP-2
To determine and analyze the molecular mechanisms involved in trafficking of MT1-MMP, we employed breast carcinoma MCF7 cells stably transfected with the original pcDNA3.1 plasmid (MCF-mock cells), or the pcDNA3.1 plasmid bearing the wild type (MCF-MT-WT cells). We also used mutant MT1-MMP, including the catalytically inert MT1-MMP-E240A mutant (MCF-MT-E240A cells), and the C-end truncated MT1-MMP-ΔCT mutant (MCF-MT-ΔCT cells) lacking the cytoplasmic tail peptide sequence. To evaluate cell
Discussion
ECM degradation and tissue remodeling play critical roles in malignant progression, particularly in tumor invasion, metastasis, and neovascularization [1]. It has been well established that certain proteinases from the human MMP family are essential for these complex, multistep pathological events [4]. At present, 23 individual human MMPs are identified and partially characterized [48]. Understanding the functional roles and the regulation of individual MMPs is of paramount importance to
Acknowledgements
This work was supported by NIH Grants CA83017 and CA77470, California Breast Cancer Research Program Grant 5JB0094, and Susan G. Komen Breast Cancer Foundation Grant 9849 (all to AYS).
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