Christopher M. Overall

Strategies for MMP inhibition in cancer: innovations for the post-trial era

For more than two decades, the view that tumour-associated matrix metalloproteinases (MMPs) were required for peritumour tissue degradation and metastasis dominated the drive to develop MMP inhibitors as anticancer therapeutics. Until recently, clinical trials with MMP inhibitors have yielded disappointing results, highlighting the need for better insight into the mechanisms by which this growing family of multifunctional enzymes contribute to tumour growth. It is now recognized that MMP activity is tightly regulated at several levels, providing new avenues for blocking these enzymes. What are the different approaches that can be used to target MMPs, and which of these might lead to new therapeutic strategies for cancer?

Validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy

The matrix metalloproteinases (MMPs) mediate homeostasis of the extracellular environment. They have multiple signalling activities that are commonly altered during tumorigenesis and that might serve as intervention points for anticancer drugs. However, there are many criteria to consider in validating MMPs as drug targets and for the development of MMP inhibitors. The inhibition of some MMPs could have pro-tumorigenic effects (making them anti-targets), counterbalancing the benefits of target inhibition. These effects might partially account for the failure of MMP inhibitors in clinical trials. What are the major challenges in MMP target validation and MMP-inhibitor-drug development?

Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.

Invasive cell migration through tissue barriers requires pericellular remodelling of extracellular matrix (ECM) executed by cell-surface proteases, particularly membrane-type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Using time-resolved multimodal microscopy, we show how invasive HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells coordinate mechanotransduction and fibrillar collagen remodelling by segregating the anterior force-generating leading edge containing β1 integrin, MT1-MMP and F-actin from a posterior proteolytic zone executing fibre breakdown. During forward movement, sterically impeding fibres are selectively realigned into microtracks of single-cell calibre. Microtracks become expanded by multiple following cells by means of the large-scale degradation of lateral ECM interfaces, ultimately prompting transition towards collective invasion similar to that in vivo. Both ECM track widening and transition to multicellular invasion are dependent on MT1-MMP-mediated collagenolysis, shown by broad-spectrum protease inhibition and RNA interference. Thus, invasive migration and proteolytic ECM remodelling are interdependent processes that control tissue micropatterning and macropatterning and, consequently, individual and collective cell migration.

Human and mouse proteases: a comparative genomic approach

The availability of the human and mouse genome sequences has allowed the identification and comparison of their respective degradomes — the complete repertoire of proteases that are produced by these organisms. Because of the essential roles of proteolytic enzymes in the control of cell behaviour, survival and death, degradome analysis provides a useful framework for the global exploration of these protease-mediated functions in normal and pathological conditions.

Loss of collagenase-2 confers increased skin tumor susceptibility to male mice

Matrix metalloproteinases (MMPs) have fundamental roles in tumor progression, but most clinical trials with MMP inhibitors have not shown improvements in individuals with cancer. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8−/−mice. Female Mmp8−/−mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.

Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites.

The function of ancillary domains and modules attached to the catalytic domain of mutidomain proteases, such as the matrix metalloproteinases (MMPs), are not well understood. The importance of discrete MMP substrate binding sites termed exosites on domains located outside the catalytic domain was first demonstrated for native collagenolysis. The essential role of hemopexin carboxyl-domain exosites in the cleavage of noncollagenous substrates such as chemokines has also been recently revealed. This article updates a previous review of the role of substrate recognition by MMP exosites in both preparing complex substrates, such as collagen, for cleavage and for tethering noncollagenous substrates to MMPs for more efficient proteolysis. Exosite domain interaction and movements—“molecular tectonics”—that are required for native collagen triple helicase activity are discussed. The potential role of collagen binding in regulating MMP-2 (gelatinase A) activation at the cell surface reveals unexpected consequences of substrate interactions that can lead to collagen cleavage and regulation of the activation and activity of downstream proteinases necessary to complete the collagenolytic cascade.

Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors.

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.

Matrix metalloproteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics.

The biological roles of the matrix metalloproteinases (MMPs) have been traditionally associated with the degradation and turnover of most of the components of the extracellular matrix (ECM). This functional misconception has been used for years to explain the involvement of the MMP family in developmental processes, cell homeostasis and disease, and led to clinical trials of MMP inhibitors for the treatment of cancer that failed to meet their endpoints and cast a shadow on MMPs as druggable targets. Accumulated evidence from a great variety of post-trial MMP degradomics studies, ranging from transgenic models to recent state-of-the-art proteomics screens, is changing the dogma about MMP functions. MMPs regulate cell behavior through finely tuned and tightly controlled proteolytic processing of a large variety of signaling molecules that can also have beneficial effects in disease resolution. Moreover, net proteolytic activity relies upon direct interactions between the different protease and protease inhibitor families, interconnected in a complex protease web, with MMPs acting as key nodal components. Such complexity renders simple interpretation of Mmp knockout mice very difficult. Indeed, the phenotype of these models reveals the response of a complex system to the loss of one protease rather than necessarily a direct effect of the lack of functional activity of a protease. Such a shift in the MMP functional paradigm, together with the difficulties associated with current methods of studying proteases this highlights the need for new high content degradomics approaches to uncover and annotate MMP activities in vivo and identify novel interactions within the protease web. Integration of these techniques with specifically designed animal models for final validation should lay the foundations for the development of new inhibitors that specifically target disease-related MMPs and/or their upstream effectors that cause deleterious effects in disease, while sparing MMP functions that are protective.

HIV-induced metalloproteinase processing of the chemokine stromal cell derived factor-1 causes neurodegeneration

The mechanisms of neurodegeneration that result in human immunodeficiency virus (HIV) type 1 dementia have not yet been identified. Here, we report that HIV-infected macrophages secrete the zymogen matrix metalloproteinase-2 (MMP-2), which is activated by exposure to MT1-MMP on neurons. Stromal cell–derived factor 1α (SDF-1), a chemokine overexpressed by astrocytes during HIV infection, was converted to a highly neurotoxic protein after precise proteolytic processing by active MMP-2, which removed the N-terminal tetrapeptide. Implantation of cleaved SDF-1(5–67) into the basal ganglia of mice resulted in neuronal death and inflammation with ensuing neurobehavioral deficits that were abrogated by neutralizing antibodies to SDF-1 and an MMP inhibitor drug. Hence, this study identifies a new in vivo neurotoxic pathway in which cleavage of a chemokine by an induced metalloproteinase results in neuronal apoptosis that leads to neurodegeneration.

In search of partners: linking extracellular proteases to substrates

Proteases function as molecular switches in signalling circuits at the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which proteases cleave a particular substrate and which substrates individual proteases cleave. Elucidating the substrate degradomes of proteases will help us to understand the function of proteases in development and disease and to validate proteases as drug targets.