Stephen J. Weiss

EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells

The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. Here we investigate the functional role of EZH2 in cancer cell invasion and breast cancer progression. EZH2 transcript and protein were consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation.

Matrix Metalloproteinases Regulate Neovascularization by Acting as Pericellular Fibrinolysins

During angiogenesis, endothelial cells penetrate fibrin barriers via undefined proteolytic mechanisms. We demonstrate that the fibrinolytic plasminogen activator (PA)-plasminogen system is not required for this process, since tissues isolated from PA- or plasminogen-deficient mice successfully neovascularize fibrin gels. By contrast, neovessel formation, in vitro and in vivo, is dependent on fibrinolytic, endothelial cell-derived matrix metalloproteinases (MMP). MMPs directly regulate this process as invasion-incompetent cells penetrate fibrin barriers when transfected with the most potent fibrinolytic metalloproteinase identified in endothelium, membrane type-1 MMP (MT1-MMP). Membrane display of MT1-MMP is required, as invasion-incompetent cells expressing a fibrinolytically active, transmembrane-deleted form of MT1-MMP remain noninvasive. These observations identify a PA-independent fibrinolytic pathway wherein tethered MMPs function as pericellular fibrinolysins during the neovascularization process.

Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix.

Cancer cells are able to proliferate at accelerated rates within the confines of a three-dimensional (3D) extracellular matrix (ECM) that is rich in type I collagen. The mechanisms used by tumor cells to circumvent endogenous antigrowth signals have yet to be clearly defined. We find that the matrix metalloproteinase, MT1-MMP, confers tumor cells with a distinct 3D growth advantage in vitro and in vivo. The replicative advantage conferred by MT1-MMP requires pericellular proteolysis of the ECM, as proliferation is fully suppressed when tumor cells are suspended in 3D gels of protease-resistant collagen. In the absence of proteolysis, tumor cells embedded in physiologically relevant ECM matrices are trapped in a compact, spherical configuration and unable to undergo changes in cell shape or cytoskeletal reorganization required for 3D growth. These observations identify MT1-MMP as a tumor-derived growth factor that regulates proliferation by controlling cell geometry within the confines of the 3D ECM.

Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells.

Human neutrophils stimulated with phorbol myristate acetate were able to destroy suspensions or monolayers of cultured human endothelial cells. Neutrophil-mediated cytotoxicity was related to phorbol myristate acetate concentration, time of incubation and neutrophil number. Cytolysis was prevented by the addition of catalase, while superoxide dismutase had no effect on cytotoxicity. The addition of the heme-enzyme inhibitors, azide or cyanide, markedly stimulated neutrophil-mediated damage while exogenous myeloperoxidase failed to stimulate cytolysis. Neutrophils isolated from patients with chronic granulomatous disease did not destroy the endothelial cell targets while myeloperoxidase-deficient neutrophils successfully mediated cytotoxicity. Endothelial cell damage mediated by the myeloperoxidase deficient cells was also inhibited by catalase but not superoxide dismutase. The addition of purified myeloperoxidase to the deficient cells did not stimulate cytotoxicity. Glucose-glucose oxidase, an enzyme system capable of generating hydrogen peroxide, could replace the neutrophil as the cytotoxic mediator. The addition of myeloperoxidase at low concentrations of glucose oxidase did not increase cytolysis, but at the higher concentrations of glucose oxidase it stimulated cytotoxicity. The destruction of endothelial cells by the glucose oxidase-myeloperoxidase system was inhibited by the addition of hypochlorous acid scavengers. In contrast, neutrophil-mediated cytolysis was not effectively inhibited by the hypochlorous acid scavengers. Based on these observations, we propose that human neutrophils can destroy cultured human endothelial cells by generating cytotoxic quantities of hydrogen peroxide.

Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP

As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from gene-targeted mice, a matrix metalloproteinase (MMP)–dependent activity is identified that drives invasion independently of plasminogen, the gelatinase A/TIMP-2 axis, gelatinase B, collagenase-3, collagenase-2, or stromelysin-1. In contrast, deleting or suppressing expression of the membrane-tethered MMP, MT1-MMP, in fibroblasts or tumor cells results in a loss of collagenolytic and invasive activity in vitro or in vivo. Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.

Protease-dependent versus-independent cancer cell invasion programs: Three-dimensional amoeboid movement revisited

Tissue invasion during metastasis requires cancer cells to negotiate a stromal environment dominated by cross-linked networks of type I collagen. Although cancer cells are known to use proteinases to sever collagen networks and thus ease their passage through these barriers, migration across extracellular matrices has also been reported to occur by protease-independent mechanisms, whereby cells squeeze through collagen-lined pores by adopting an ameboid phenotype. We investigate these alternate models of motility here and demonstrate that cancer cells have an absolute requirement for the membrane-anchored metalloproteinase MT1-MMP for invasion, and that protease-independent mechanisms of cell migration are only plausible when the collagen network is devoid of the covalent cross-links that characterize normal tissues.

A Wnt–Axin2–GSK3β cascade regulates Snail1 activity in breast cancer cells

Accumulating evidence indicates that hyperactive Wnt signalling occurs in association with the development and progression of human breast cancer. As a consequence of engaging the canonical Wnt pathway, a β-catenin–T-cell factor (TCF) transcriptional complex is generated, which has been postulated to trigger the epithelial–mesenchymal transition (EMT) that characterizes the tissue-invasive phenotype. However, the molecular mechanisms by which the β-catenin–TCF complex induces EMT-like programmes remain undefined. Here, we demonstrate that canonical Wnt signalling engages tumour cell dedifferentiation and tissue-invasive activity through an Axin2-dependent pathway that stabilizes the Snail1 zinc-transcription factor, a key regulator of normal and neoplastic EMT programmes. Axin2 regulates EMT by acting as a nucleocytoplasmic chaperone for GSK3β, the dominant kinase responsible for controlling Snail1 protein turnover and activity. As dysregulated Wnt signalling marks a diverse array of cancerous tissue types, the identification of a β-catenin–TCF-regulated Axin2–GSK3β–Snail1 axis provides new mechanistic insights into cancer-associated EMT programmes.

Transmembrane-deletion Mutants of the Membrane-type Matrix Metalloproteinase-1 Process Progelatinase A and Express Intrinsic Matrix-degrading Activity

Membrane-type matrix metalloproteinase-1 (MT-MMP-1) has been proposed to play a critical role in regulating the expression of tissue-invasive phenotypes in normal and neoplastic cells by directly or indirectly mediating the activation of progelatinase A. To begin characterizing MT-MMP-1 structure-function relationships, transmembrane-deletion mutants were constructed, and the processing of the zymogens as well as the enzymic activity of the mature proteinases was analyzed. We now demonstrate that pro-MT-MMP-1 mutants are efficiently processed to active proteinases following post-translational endoproteolysis immediately downstream of an Arg-Arg-Lys-Arg basic motif by a proprotein convertase-dependent pathway. The secreted form of active MT-MMP-1 not only displays an N terminus identical with that described for the processed wild-type enzyme at Tyr (Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grants, G. A., and Goldberg, G. I.(1995) J. Biol. Chem. 270, 5331-5338), but also directly mediated progelatinase A activation via a two-step proteolytic cascade indistinguishable from that observed with intact cells. Furthermore, although the only function previously ascribed to MT-MMP-1 is its ability to act as a progelatinase A activator, purified transmembrane deletion mutants also expressed proteolytic activities against a wide range of extracellular matrix molecules. Given recent reports that MT-MMP-1 ectodomains may undergo intercellular transfer in vivo (Okada, A., Bellocq, J.-P., Rouyer, N., Chenard, M.-P., Rio, M.-C., Chambon, P., and Basset, P.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2730-2734), our data suggest that soluble forms of the proteinase confer recipient cells with the ability to not only process progelatinase A, but also directly degrade extracellular matrix components.

Breaching the basement membrane: who, when and how?

The basement membrane (BM), a specialized network of extracellular matrix macromolecules, surrounds epithelial, endothelial, muscle, fat and nerve cells. During development, immune surveillance and disease states ranging from cancer to fibrosis, host cells penetrate the BM by engaging tissue-invasive programs, the identity of which remain largely undefined. Although it is commonly assumed that all cells employ similar mechanisms to cross BM barriers, accumulating evidence indicates that cells might selectively mobilize protease-dependent or -independent invasion programs. New data indicate that protease-dependent transmigration is largely reliant on a group of membrane-anchored metalloenzymes, termed the membrane-type matrix metalloproteinases, which irreversibly remodel BM structure. By contrast, mechanisms that enable protease-independent transmigration remain undefined and potentially involve the reversible disassembly of the BM network. Further characterization of the molecular mechanisms underlying BM transmigration should provide important insights into pathophysiologic tissue remodeling events and also enable the development of novel therapeutics.

A Pericellular Collagenase Directs the 3-Dimensional Development of White Adipose Tissue

White adipose tissue (WAT) serves as the primary energy depot in the body by storing fat. During development, fat cell precursors (i.e., preadipocytes) undergo a hypertrophic response as they mature into lipid-laden adipocytes. However, the mechanisms that regulate adipocyte size and mass remain undefined. Herein, we demonstrate that the membrane-anchored metalloproteinase, MT1-MMP, coordinates adipocyte differentiation in vivo. In the absence of the protease, WAT development is aborted, leaving tissues populated by mini-adipocytes which render null mice lipodystrophic. While MT1-MMP preadipocytes display a cell autonomous defect in vivo, null progenitors retain the ability to differentiate into functional adipocytes during 2-dimensional (2-D) culture. By contrast, within the context of the 3-dimensional (3-D) ECM, normal adipocyte maturation requires a burst in MT1-MMP-mediated proteolysis that modulates pericellular collagen rigidity in a fashion that controls adipogenesis. Hence, MT1-MMP acts as a 3-D-specific adipogenic factor that directs the dynamic adipocyte-ECM interactions critical to WAT development.