Matrix metalloproteinases: regulators of the tumor microenvironment

Abstract

Extracellular proteolysis mediates tissue homeostasis. In cancer, altered proteolysis leads to unregulated tumor growth, tissue remodeling, inflammation, tissue invasion, and metastasis. The matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis. Recent technological developments have markedly advanced our understanding of MMPs as modulators of the tumor microenvironment. In addition to their role in extracellular matrix turnover and cancer cell migration, MMPs regulate signaling pathways that control cell growth, inflammation, or angiogenesis and may even work in a nonproteolytic manner. These aspects of MMP function are reorienting our approaches to cancer therapy.

Figure 1. MMP Composition and Expression in the Stroma

(A) Matrix metalloproteinases (MMPs) are comprised of different subdomains. All MMPs have the “minimal domain” in common, which contains three principal regions: an amino-terminal signal sequence (Pre) to be cleaved by the signal peptidase during entry into the endoplasmic reticulum, a pro-domain (Pro) containing a thiol-group (-SH) and a furin cleavage site, and the catalytic domain with a zinc-binding site (Zn²⁺). Interaction of the -SH group of the pro-domain with the zinc ion of the catalytic domain keeps the enzyme as an inactive zymogen. Activation of the zymogen is often mediated by intracellular furin-like proteinases that target the furin recognition motif (Fu) between the pro-domain and the catalytic domain. In addition to the minimal domain, most MMPs possess a hemopexin-like region, a domain composed of four repeats that resemble hemopexin and contain a disulfide bond (S-S) between the first and the last subdomain, which is linked to the catalytic domain via a flexible hinge region. Besides their differential domain structure, MMPs can be principally divided into secreted (MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -19, -20, -21, -22, -27, -28) and membrane-anchored proteinases (MMP-14, -15, -16, -17, -23, -24, -25), the latter of which use either a transmembrane domain (TM) with a cytoplasmic domain (Cy) attached to it, a glycosylphosphatidylinositol (GPI) anchor, or an amino-terminal signal anchor (SA), which is only the case for MMP-23, as it is anchored in the plasma membrane. MMP-23 also contains the unique cysteine array (CA) and an immunoglobulin (Ig)-like domain. The gelatinases MMP-2 and -9 show gelatin-binding repeats that resemble the collagen-binding type II motif of fibronectin (FN). (B) Expression pattern of proteinases and their physiological inhibitors in non-malignant stromal cells. Cells commonly found in the microenvironment of many cancers include inflammatory cells (such as neutrophils, macrophages, dendritic cells, lymphocytes, and mast cells), endothelial cells, fibroblasts, and hematopoietic progenitor cells. These cells express a plethora of proteinases that are released into the extracellular space and influence multiple events of tumor progression. Selected examples of proteinases and endogenous inhibitors expressed by these cell types are shown.

Figure 2. Proteolytic Cascades Regulate MMP Function

MMPs are synthesized as inactive zymogens that need to be activated by proteolytic removal of the pro-domain (for instance, as carried out by plasmin). Several MMPs exert an autocrine feedback by degrading several physiological proteinase inhibitors that inhibit proteolytic conversion of MMPs including α1-chymotrypsin (α1-CT), α1-proteinase inhibitor (α1-PI), and α2-antiplasmin (α2-AP) (1). Some pro-MMPs can also be converted by other MMPs. Selected examples for mutual MMP conversion are given (2). Another physiological inhibitor, α2-macroglobulin (α2-MG), normally inhibits MMP activity but can also be degraded by several MMPs, which then prolongs MMP function (3). Inflammatory cells frequently infiltrate the tumor microenvironment and produce large amounts of reactive oxygen species (ROS), which may promote MMP activation via oxidation of the pro-domain cysteine. Myeloperoxidase (MPO) of infiltrating neutrophil catalyzes the transformation of ROS into hypochloric acid (HOCl), which may interfere with MMP activity by chemical modification of crucial residues of the catalytic domain (4). On the other hand, active MMPs may also launch a negative feedback, for instance, by degrading plasminogen and therefore prohibiting the conversion into MMP-activating plasmin (5). The complex interaction of proteinases and their inhibitors under physiological circumstances happens upstream of the physiological functions of MMPs, such as matrix remodeling, angiogenesis, cellular signaling, and cancer cell migration.

Figure 3. Multiple Functions of MMPs in the Tumor Microenvironment

Tumor progression and metastasis involve different stages, all of which can be modulated by MMPs and other extracellular proteinases. MMPs are mainly provided by nonmalignant, infiltrating stromal cells such as neutrophils, macrophages, or endothelial cells. Selected examples of proteinases and their target substrates in each of these steps are given in numbered boxes. Tissue invasion of the tumor (1) and cancer cell intravasation into blood vessels (2) require extracellular matrix (ECM) remodeling and downregulation of cellular adhesion. MMPs, ADAMs, and other proteinases such as cathepsins (Cat)-B, -K, and -L are implicated in the turnover of ECM components, but they also regulate cancer cell migration, for example by cleaving proteinase-activated receptor (PAR)-1 or by degrading cell surface molecules that mediate cellular adhesion, such as CD44 or E-cadherin. The egress of metastatic tumor cells into the circulation is often directly accompanied by tumor-associated macrophages and may exploit proteolytic functions that mediate leukocyte migration across the endothelium and the endothelial basement membrane (EBM) under physiological conditions (2). Tumors are highly vascularized tissues and the formation of new blood vessels (angiogenesis; 3) can be triggered by the release of vascular endothelial growth factor (VEGF), which is mainly facilitated by MMP-2 and -9. Moreover, MMPs may also regulate angiogenesis by the generation of angiostatin-like peptides through the cleavage of plasminogen (Plg). MMPs are potent regulators of inflammation (4), thus they are critically involved in the recruitment of inflammatory cells to the tumor microenvironment, for example by converting TNF-α or interleukin-8 (IL-8). They also generate chemotactic peptides such as PGP through the degradation of collagen (col) and form chemotactic gradients by cleaving the ECM component syndecan to release soluble gradients of CXCL1/KC, a potent neutrophil-attracting chemokine. Some MMPs exert anti-inflammatory function, for example by degrading monocyte chemotactic protein (MCP3/CCL7). Metastasis results in the dissemination of malignant cells to secondary sites distant to the primary tumor. Recent findings indicate that these distant sites may be primed for metastasis by inflammatory cells and hematopoietic progenitor cells (HPCs) that locate to these sites to form a so-called premetastatic niche (5). MMP-9 and MMP-2 are involved in this process most likely by releasing factors such as VEGF and Kit ligand (Kit-L), which recruits HPCs from the bone marrow (BM).

Figure 4. Modulation of the Tumor Microenvironment by MMPs

Summary of the various processes that are modulated by MMPs in the tumor microenvironment. The selected examples of MMPs and ADAMs promote (pro) or suppress (anti) these processes. An intravital microscopy image of the mammary gland of MMTV-PyMT mice that spontaneously develop mammary carcinoma was taken using a spinning disc inverted confocal microscope (Egeblad et al., 2008). These mice also express enhanced cyan fluorescent protein (CFP) under the control of the actin promoter (ACTB-ECFP) to enable tumor cell labeling (blue) and enhanced green fluorescent protein (GFP) expression under the control of a c-fms promoter (c-fms-EGFP) to label myeloid cells (green). These mice were injected intravenously with 70 kDa rhodamine-dextran to visualize blood vessels (red). This image illustrates the complexity of the tumor microenvironment, which is largely influenced by nonmalignant cells, such as myeloid cells, all of which could be targets as well as sources for MMPs.