EMMPRIN, a Tumor Cell Surface Inducer of Matrix Metalloproteinase Production in Stromal Cells
EMMPRIN, a Tumor Cell Surface Inducer of Matrix Metalloproteinase Production in Stromal Cells
By Bryan P. Toole, PhD, and Stanley Zucker, MD
One of the crucial processes in tumorigenesis is proteolytic modification of the pericellular matrix surrounding tumor cells by matrix metalloproteinases (MMPs), a large family of zinc dependent endopeptidases.1 MMPs have been implicated in several steps in tumor progression, including invasion though basement membranes and interstitial matrices, angiogenesis, and tumor cell growth. Strong support for the involvement of MMPs at some step in tumor progression comes from experiments in which tissue inhibitors of MMPs (TIMPs) or synthetic inhibitors of metalloproteinases have been shown to prevent tumor growth and metastasis in experimental animal models.2,3 The classic three-step hypothesis of cancer cell invasion involves attachment of the cancer cells to basal laminae and local degradation of this matrix by MMPs, followed by tumor cell locomotion.4 Current evidence now suggests that MMPs are important in tumor growth, e.g. by processing growth factor precursors, as well as in mediating cell migration and invasion.1,4-7
Stromal Production of Tumor MMPs
A surprising development in this field over the past several years has been the growing awareness that, in vivo, most tumor MMPs, such as interstitial collagenase (MMP-1), collagenase-3 (MMP-13), gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin-1 (MMP-3), stromelysin-3 (MMP-11), and membrane type-MMPs (MT-MMPs), are produced by stromal cells associated with tumors rather than by (or in addition to) the tumor cells themselves.8,9 Moreover, it appears from recent data that stromal MMPs contribute significantly to tumor progression in vivo.9-11 In some cases, however, a given MMP may be produced by stromal cells or tumor cells at different stages of progression. Matrilysin (MMP-7) appears to be unique in its restriction to epithelial and carcinoma cells.
The almost ubiquitous production of MMPs by stromal cells within tumors, but not within most normal adult tissues, implies that tumor cells exert regulatory effects on the stromal cells, inducing them to express elevated levels of MMPs. Although it is clear that soluble cytokines contribute to this process, it is also apparent that tumor cell membrane-bound factors are involved.
The first systematic investigation of the latter took place in the laboratory of Dr. Chitra Biswas, where initial experiments suggested that tumor cell-secreted or shed factors were responsible for stimulation of new synthesis of interstitial collagenase (MMP-1) by fibroblasts.12 However, subsequent experiments showed that the source of most of the MMP-1-stimulatory factor produced by B16 murine melanoma and LX-1 human lung carcinoma cells was plasma membrane-derived, and that this factor could act via direct cell-cell interaction or via shedding of the factor from the cell surface. Subsequently, the factor (originally called tumor cell-derived collagenase stimulatory factor [TCSF]) was shown to stimulate production of several MMPs by fibroblasts and was fully characterized; it was shown to be a transmembrane glycoprotein and a member of the Ig superfamily, and was renamed extracellular matrix metalloproteinase inducer (EMMPRIN).13-16 Tragically, Dr. Biswas died in 1993, after having completed the characterization of EMMPRIN. Subsequently, her collaborators have continued her pioneering work.
EMMPRIN, a Tumor Cell Surface Inducer of MMP Production by Tumor Stromal Cells
On cloning of EMMPRIN cDNA15, it became apparent that EMMPRIN is identical to human basigin and the M6 antigen present on membranes of leukocytes from patients with arthritis;17,18 the functions of these proteins were not then known. EMMPRIN/basigin/M6 (now also CD147) is homologous to proteins independently discovered in a wide variety of systems in other species, but in each case they were of unknown function at that time.17-21
It is also evident that there is a family of molecules related to EMMPRIN (e.g., rat synaptic membranes contain two major Ig superfamily proteins, gp65 and gp55) that are closely related but not identical to the rat homolog of EMMPRIN.22 Structural analyses demonstrated that the transmembrane domain of EMMPRIN is almost perfectly conserved among species, suggesting that this region is of functional importance. The properties of the transmembrane region also suggested that intramembrane interactions with other proteins are likely to occur, and it has now been shown that EMMPRIN interacts with the integrins, a3b1 and a6b1, within the plasma membrane of HT1080 fibrosarcoma cells.17,18,23 Recently, a knockout mouse has been produced in which basigin, the murine homolog of EMMPRIN, is lacking.24 The null mutant is in most cases unable to undergo implantation, presumably due to the involvement of MMPs in this process.25 However some embryos implant and survive past birth, but they are sterile and have deficiencies in spermatogenesis, sensory and memory functions, and lymphocyte responses.24 The relevance of MMP stimulation to these latter processes has not been established.
Although it is now apparent that many embryonic and adult tissues express EMMPRIN, the level of EMMPRIN expression in tumors, especially malignant tumors, is usually very much greater than in corresponding normal tissue.26-30 For example, in one study, the relative distribution of EMMPRIN and MMP-2 mRNAs was compared by in situ hybridization in normal lung tissue vs. squamous cell carcinomas of the lung and in benign mammary growths vs. ductal carcinomas of the breast.27 EMMPRIN mRNA was detected in all breast carcinomas and the majority of lung carcinomas. Both pre-invasive and invasive cancer cells were positive, but tumor stromal cells and peritumoral tissue showed insignificant EMMPRIN mRNA reactivity. Normal and benign epithelia were negative. MMP-2 and MMP-1 mRNAs, on the other hand, were restricted to stromal cells close to tumor clusters.27,28 The expression of EMMPRIN mRNA was also analyzed by Northern blots, which were then densitometrically scanned; the results showed insignificant expression in normal or benign tissues but high levels at all stages of tumor progression in both lung and breast tumors. Analyses of distribution within tumors were also made by quantitative image cytometry, and showed that near equivalently high levels of EMMPRIN mRNA were expressed in pre-invasive and invasive nests of tumor cells vs. insignificant amounts in normal or peritumoral tissues.27 Both normal and tumor epithelia stained positively with antibody to EMMPRIN, but expression of EMMPRIN was much stronger in tumor tissue.29 In another study, EMMPRIN levels were shown to be much higher in transitional cell carcinomas of the bladder than in normal bladder epithelium.26 Although EMMPRIN is expressed at a moderately high level in normal non-neoplastic keratinocytes, its expression in oral squamous cell carcinoma promotes MMP production and tumor cell invasion.31,32
EMMPRIN and Tumor Progression
Since malignant tumor cells consistently express EMMPRIN in vivo and in vitro at much higher levels than normal and benign cells and tissues, we recently tested whether over-expression of EMMPRIN stimulates tumor progression.33 We used human breast carcinoma cells that produce slow-growing primary tumors in nude mice and express relatively low levels of EMMPRIN. We transfected the cells with EMMPRIN or with vector alone, and selected stable transfectant clones. Increased expression of EMMPRIN in the selected EMMPRIN transfectants was demonstrated by Northern blotting and immunocytology. The transfectants grew at similar rates to wild-type cells in culture.
In three separate in vivo experiments with different transfectant clones, the cells were injected into the mammary fat pad of groups of 10 nu/nu, athymic, female nude mice. In all three experiments, the mice injected with EMMPRIN transfectants grew large tumors over a 12-week period, whereas controls grew small tumors that were primarily detectable only at autopsy. In addition, the EMMPRIN transfectants consistently gave rise to much more extensive invasion into surrounding abdominal wall muscle than controls. Mouse survival was markedly decreased with the EMMPRIN transfectants vs. controls.33 EMMPRIN may also participate in angiogenic mechanisms since it stimulates MMP production by endothelial cells.
Docking of MMPs to the Tumor Cell Surface
After synthesis and secretion of MMPs by stromal cells, some of these enzymes have been shown to bind back to the tumor cell surface. For example, MMP-2 binds to either alphaV-beta3 integrin or to a TIMP2-MT-MMP complex.34-36 On binding to the latter, MMP-2 is activated by a second, free MT-MMP molecule. This activation cascade is also stimulated by EMMPRIN.37 A similar mechanism of binding and activation involving MT-MMP appears to occur with collagenase-3. Gelatinase B can be bound to the cell surface via CD44 or via a component of collagen IV.6,38 Presentation of MMPs at these docking sites has been shown to promote tumor cell invasiveness.6,34,35 In a recent study from our laboratories we have shown that, in addition to stimulating MMP production, EMMPRIN may be a docking protein for MMP-1 on the tumor cell surface.39 This study showed by phage display, affinity chromatography, and immunocytochemistry that MMP-1 forms a complex with EMMPRIN on the surface of human lung carcinoma cells. Since collagenase activity is essential for invasion of fibrous tissues, localization of MMP-1 on the tumor cell surface would facilitate this process.40
Conclusions
The stromal response to tumors has heretofore been well known to surgical pathologists, but has received scant attention from the scientific community. We propose that activated fibroblasts, serving as drones of tumor cells, are important components of the cancer growth and invasive processes. It is becoming increasingly apparent that tumor cells create a pericellular environment in which many stromal cell-produced MMPs and other proteases become concentrated, thereby enhancing the ability of tumor cells to invade extracellular matrices and locally to process precursors of factors that promote tumor growth. EMMPRIN appears to play a central role in these processes in that it stimulates stromal MMP synthesis and MMP-2 activation, and is a docking protein for MMP-1 on the tumor cell surface.
We are currently attempting to design and produce antagonists of EMMPRIN-receptor interaction that could be used for therapeutic manipulation of EMMPRIN function. (Dr. Toole is Professor of Anatomy and Cellular Biology, Tufts Medical School, Boston, MA, and Dr. Zucker is Professor of Medicine, Departments of Research and Medicine, Veteran Affairs Medical Center, Northport, NY.)
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Matrilysin (MMP-7) appears to be unique in that it is produced exclusively by:
a. fibroblasts.
b. mesenchymal cells.
c. epithelial and carcinoma cells.
d. stroma.
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