CD73 complexes with emmprin to regulate MMP-2 production from co-cultured sarcoma cells and fibroblasts.

BACKGROUND: Interaction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts. METHODS: CD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays. RESULTS: Stimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells. CONCLUSION: This study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.

Cooperative roles for emmprin and LYVE-1 in the regulation of chemoresistance for primary effusion lymphoma.

The Kaposi's sarcoma-associated herpesvirus is the causative agent of primary effusion lymphoma (PEL), for which cytotoxic chemotherapy represents the standard of care. The high mortality associated with PEL may be explained in part by resistance of these tumors to chemotherapy. The membrane-bound glycoprotein emmprin (CD147) enhances chemoresistance in tumors through effects on transporter expression, trafficking and interactions. Interactions between hyaluronan and hyaluronan receptors on the cell surface also facilitate emmprin-mediated chemoresistance. Whether emmprin or hyaluronan-receptor interactions regulate chemotherapeutic resistance for virus-associated malignancies is unknown. Using human PEL tumor cells, we found that PEL sensitivity to chemotherapy is directly proportional to expression of emmprin, the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and a drug transporter known as the breast cancer resistance protein/ABCG2 (BCRP), and that emmprin, LYVE-1 and BCRP interact with each other and colocalize on the PEL cell surface. In addition, we found that emmprin induces chemoresistance in PEL cells through upregulation of BCRP expression, and RNA interference targeting of emmprin, LYVE-1 or BCRP enhances PEL cell apoptosis induced by chemotherapy. Finally, disruption of hyaluronan-receptor interactions using small hyaluronan oligosaccharides reduces expression of emmprin and BCRP while sensitizing PEL cells to chemotherapy. Collectively, these data support interdependent roles for emmprin, LYVE-1 and BCRP in chemotherapeutic resistance for PEL.

Extracellular matrix metalloproteinase inducer regulates metalloproteinases in human uterine endometrium.

CONTEXT: Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. OBJECTIVE: The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). MAIN OUTCOME MEASURES: EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. RESULTS: EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. CONCLUSIONS: The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.

Tumorigenic potential of extracellular matrix metalloproteinase inducer.

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs.

Hyaluronan in morphogenesis.

Hyaluronan is a very large polysaccharide that is found in extracellular matrices, at the cell surface and inside cells. This review focuses on the functions of hyaluronan directly associated with the cell surface, where it is commonly present as the essential core of a highly hydrated pericellular matrix that contains several other components (hyaladherins) bound to hyaluronan. Three major molecular characteristics of hyaluronan contribute to its physiological functions: its unique hydrodynamic properties, its interactions with structural extracellular hyaladherins, and its instructive effects on cell signaling and behavior. Recent studies of hyaluronan-deficient mouse embryos illustrate the importance of each of these classes of function of hyaluronan. It is postulated that the morphogenetic effects of hyaluronan are due to its ability to act as a template for assembly of a multi-component, pericellular matrix as well as to its physical properties. This matrix would provide a hydrated environment in which cells are separated from structural barriers to morphogenetic changes and receive signals from hyaluronan itself and from associated factors.

Basigin (murine EMMPRIN) stimulates matrix metalloproteinase production by fibroblasts.

Analysis of basigin-null mice has shown that basigin is involved in several important physiological processes including reproductive, immune, and neural activities (Igakura et al., 1998, Dev Biol 194:152-165). However, its molecular mechanism of action in these processes has not yet been established. Our objective here is to determine whether basigin has functional properties similar to its apparent human tumor cell homolog, EMMPRIN, i.e., the ability to stimulate matrix metalloproteinase (MMP) production in fibroblasts (Guo et al. 1997, J Biol Chem 272:24-27). Mouse cells express two major forms of basigin that differ in their degree of glycosylation (molecular weights: 45 and 58 kDa) but, in similar fashion to human EMMPRIN, mouse tumor cells express higher levels of basigin than normal cells. We have used three different methods to show that basigin stimulates MMP expression in fibroblasts. First, recombinant basigin was partially purified from transfected CHO cells by affinity chromatography. This basigin preparation stimulates production of MMPs on addition to fibroblasts in culture. Second, co-culture of basigin-transfected CHO cells with fibroblasts gives rise to increased expression of MMPs as compared to control co-cultures. Third, we employed a novel approach in which a recombinant basigin adenovirus was constructed and used to infect the target fibroblasts, so that mutual stimulation between neighboring fibroblasts would be expected to result. In this method also, basigin stimulates production of MMPs. Finally, we showed that addition of basigin or EMMPRIN antibody, respectively, to recombinant basigin or EMMPRIN adenovirus-infected cells augments stimulation of MMP synthesis, implying that cross-linking of basigin/EMMPRIN in the membrane enhances activity. We conclude that murine basigin and human EMMPRIN have similar MMP-inducing activities and are functional homologs.

Expression of emmprin (CD147), a cell surface inducer of matrix metalloproteinases, in normal human brain and gliomas.

EMMPRIN (extracellular matrix metalloproteinase inducer), also called CD147, basigin or M6 in the human, is a member of the immunoglobulin superfamily that is present on the surface of tumor cells and stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPs). In our study, we investigated expression of EMMPRIN in human normal brain and gliomas, since mouse basigin and chicken HT7, the species homologues of human EMMPRIN, are associated with neuronal interactions and normal blood-brain barrier function, respectively. EMMPRIN expression was detected in all samples of non-neoplastic brain and glioma tissues examined. However, expression levels of EMMPRIN mRNA and protein were significantly higher in gliomas than in non-neoplastic brain. Moreover, levels of mRNA expression and immunohistochemical staining correlated with tumor progression in gliomas: They were highest in the most malignant form of glioma, glioblastoma multiforme, followed by anaplastic astrocytoma and then low-grade astrocytoma. Also, immunolocalization revealed quite different distributions in non-neoplastic brain and glioma: EMMPRIN was demonstrated only in vascular endothelium in non-neoplastic regions of the brain, whereas it was present in tumor cells but not in proliferating blood vessels in malignant gliomas. These data indicate that an MMP inducer molecule EMMPRIN is differently expressed in human normal brain and gliomas and could be associated with astrocytoma progression. Possible mechanisms whereby glioma cell EMMPRIN could influence tumor progression will be discussed.

Glioma cell extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147) stimulates production of membrane-type matrix metalloproteinases and activated gelatinase A in co-cultures with brain-derived fibroblasts.

Extracellular matrix metalloproteinase inducer (EMMPRIN) also called CD147, basigin or M6 in the human is a member of the immunoglobulin superfamily that is enriched on the surface of tumor cells and stimulates adjacent stromal cells to produce several matrix metalloproteinases (MMPs). In this study, we have demonstrated that coculturing of EMMPRIN-expressing human glioblastoma multiforme cells (U251) with brain-derived human fibroblasts not only stimulates production, but also activation of pro-gelatinase A (proMMP-2), an enzyme that is enriched in malignant gliomas and most likely crucial to tumor progression. Production of membrane types 1 and 2-MMPs (MT1-MMP and MT2-MMP), which are activators of proMMP-2, was also stimulated in these cocultures. Stimulation of MMP-2, MT1-MMP and MT2-MMP production was inhibited by anti-EMMPRIN monoclonal antibody in a dose-dependent manner. Thus, we have shown, for the first time, that EMMPRIN causes increased expression of MT1-MMP and MT2-MMP, as well as increased production and activation of MMP-2.

Molecular characterization of a novel intracellular hyaluronan-binding protein.

Hyaluronan has well defined functions in extracellular matrices and at the surface of cells. However, several studies have now shown that significant pools of hyaluronan are also present intracellularly, but its function therein is unknown. One avenue of investigation that may assist in defining the function of intracellular hyaluronan is to identify intracellular hyaluronan-binding proteins. In previous studies we identified CDC37, a cell cycle regulatory protein, using a monoclonal antibody that recognizes a novel group of hyaluronan-binding proteins. In this study, we have identified a second hyaluronan-binding protein with this antibody and characterized its properties. This protein, which we have termed IHABP4, was also found to be an intracellular and a specific hyaluronan-binding protein, containing several hyaluronan-binding motifs: (R/K)[X(7)](R/K) (where R/K denotes arginine or lysine and X denotes non-acidic amino acids). Furthermore, we have determined the gene organization of IHABP4 and cloned cDNAs for the chick, mouse, and human homologs. Comparison of the deduced chick, mouse, and human protein sequences showed that the hyaluronan-binding motifs, (R/K)[X(7)](R/K), in these sequences are conserved; both chick and mouse IHABP4 were shown directly to bind hyaluronan. Biochemical fractionation and immunofluorescent localization of epitope-tagged IHABP4 indicated that it is mainly present in the cytoplasm. These data support the possibility that intracellular hyaluronan and its binding proteins may play important roles in cell behavior.

Perturbation of hyaluronan interactions by soluble CD44 inhibits growth of murine mammary carcinoma cells in ascites.

Hyaluronan accumulates in ascites during intraperitoneal proliferation of TA3/St murine mammary carcinoma cells and at sites of their invasion of the peritoneal wall. To determine whether hyaluronan is functionally involved in these events, ascites tumor formation was compared in mice injected intraperitoneally with stable transfectants of TA3/St cells that overexpress soluble CD44, a hyaluronan-binding protein, versus in mice injected with transfectants expressing mutated soluble CD44 that does not bind hyaluronan. The soluble CD44 transfectants temporarily grew at a reduced rate within the peritoneal cavity, then went into G(1) arrest and were subsequently cleared from the peritoneum. However, transfectants overexpressing mutant soluble CD44 that does not bind hyaluronan exhibited similar ascites accumulation, growth rates, and cell-cycle profiles in vivo to wild-type and vector-transfected TA3/St cells, all of which continued to grow until the tumors became fatal. The soluble CD44-transfected TA3/St cells also failed to attach to and form tumors in the peritoneal wall. When grown in vitro in soft agar, the soluble CD44 transfectants exhibited a dramatic reduction in colony formation compared to wild-type, vector-transfected, and mutant soluble CD44-transfected TA3/St cells. Thus, perturbation of hyaluronan interactions by soluble CD44 has a direct effect on the growth characteristics of these tumor cells, leading to inhibition of anchorage-independent growth in vitro and ascites growth in vivo.

EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface.

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as basigin or CD147, is a glycoprotein that is enriched on the surface of tumor cells and stimulates production of several matrix metalloproteinases by adjacent stromal cells. In this study, we have found that EMMPRIN not only stimulates the production of interstitial collagenase (MMP-1) but also forms a complex with MMP-1 at the tumor cell surface. Complex formation was demonstrated by phage display, affinity chromatography, and immunocytochemistry. Presentation of MMP-1 complexed to EMMPRIN at the tumor cell surface may be important in modifying the tumor cell pericellular matrix to promote invasion.

Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases.

EMMPRIN (extracellular matrix metalloproteinase inducer) also known as CD147 and basigin, is a member of the immunoglobulin family that is present on the surface of tumor cells and stimulates nearby fibroblasts to synthesize matrix metalloproteinases. Using our EMMPRIN cDNA, we have isolated a cosmid clone that contains the human EMMPRIN gene. S1 analysis with a fragment of the gene clone and primer extension of the mRNA was performed to determine the transcription start site. PCR and sequence analysis have defined the exon/intron organization of the gene and show that it is highly conserved with the mouse EMMPRIN/basigin gene. About 950 bases of the 5'-flanking region were examined for transcription factor consensus binding sites, locating three SP1 sites and two AP2 sites. The transcription start site was found to be located in a CpG island. Elements in the proximal promoter region were conserved in the human and mouse genes.

Inhibition of tumor growth in vivo by hyaluronan oligomers.

One of the critical events in tumor growth and metastasis is the interaction between tumor cells and host tissue stroma, mediated by different adhesion receptor repertoires in different tumor cell types. Several lines of evidence indicate that interaction between the hyaluronan receptor CD44, expressed on tumor cells, and host tissue stromal hyaluronan can enhance growth and invasiveness of certain tumors. Disruption of CD44-hyaluronan interaction by soluble recombinant CD44 has been shown to inhibit tumor formation by lymphoma and melanoma cells transfected with CD44. Since hyaluronan is a ubiquitous glycosaminoglycan polymer from which oligosaccharides of defined size can be readily purified, we tested the ability of hyaluronan oligomers to inhibit tumor formation by subcutaneously (s.c.) injected B16F10 melanoma cells. Our results indicate that hyaluronan oligomers injected at concentrations as low as 1 mg/ml can markedly inhibit B16F10 melanoma growth, providing a potentially attractive reagent for the control of local tumor development.

Organization of the chick CDC37 gene.

CDC37 and the chaperone protein, Hsp90, form a complex that binds to several kinases, resulting in stabilization and promotion of their activity. CDC37 also binds DNA and glycosaminoglycans in a sequence-specific manner. In this study, we further characterize chick CDC37 and examine the organization of the CDC37 gene. Chick CDC37 is a approximately 50-kDa protein encoded by an mRNA of approximately 1.7 kilobases. The CDC37 gene is approximately 8.5 kilobases and contains 8 exons and 7 introns of various sizes. The presumptive promoter and 5'-flanking regions contain an E2 box and consensus binding sites for SP1, for the S8 homeodomain protein, and for two zinc finger clusters within the myeloid progenitor transcription factor, MZF1. Particularly striking is a approximately 470-base pair region composed of a highly repetitive 10-11-base pair sequence, (T/C)gCTAT(A/G)GGG(A/T) (where g represents the additional G present in the 11-base pair sequence). This region includes 15 copies of the sequence, TATGGGGA, which conforms to the DNA consensus sequence recognized by one of the zinc finger clusters in MZF1. These findings emphasize the potential importance of CDC37 in regulation of cellular behavior during tissue development and reorganization.

Hyaluronan in morphogenesis.

Pericellular matrices surrounding migrating and proliferating cells in the developing embryo, in regenerating tissues and in other dynamic cellular events, such as tumour cell invasion, are enriched in hyaluronan. In addition to contributing to the unique structure of the pericellular matrix, hyaluronan interacts with cell surface receptors, such as RHAMM and CD44. During morphogenesis, these interactions of hyaluronan with the cell surface are important in several ways. First, hyaluronan-CD44 interactions have been shown to mediate endocytic removal of hyaluronan at critical stages of embryonic development. Secondly, hyaluronan provides an appropriately hydrated, pericellular milieu that facilitates cellular invasion. Thirdly, in-vitro studies suggest strongly that interactions of hyaluronan with RHAMM or CD44 are involved in cell movement and proliferation, which are critical events in morphogenesis.

Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells.

Many of the tumor-associated matrix metalloproteinases that are implicated in metastasis are produced by stromal fibroblasts within or surrounding the tumor in response to stimulation by factors produced by tumor cells. In this study we transfected Chinese hamster ovary cells with putative cDNA for human extracellular matrix metalloproteinase inducer (EMMPRIN), a transmembrane glycoprotein that is attached to the surface of many types of malignant human tumor cells and that has previously been implicated in stimulation of matrix metalloproteinase production in fibroblasts. We show that these transfected cells synthesize EMMPRIN that is extensively post-translationally processed; this recombinant EMMPRIN stimulates human fibroblast production of interstitial collagenase, stromelysin-1, and gelatinase A (72-kDa type IV collagenase). We propose that EMMPRIN regulates matrix metalloproteinase production during tumor invasion and other processes involving tissue remodeling.

Common pattern of CD44 isoforms is expressed in morphogenetically active epithelia.

CD44 is a widely distributed cell surface glycoprotein that is expressed as many isoforms arising from a single gene by alternative splicing. An important ligand for the widespread CD44 isoform, CD44s (standard form of CD44), is hyaluronan but ligands for the numerous variant isoforms are not as well characterized. Although it has been documented that CD44 is present at critical sites and stages of morphogenesis of several organs, the nature of the isoforms expressed and their precise localization have not been described. In this study we have determined the identity and distribution of CD44 isoforms expressed during development of several embryonic mouse organs by a combination of immunohistochemistry, in situ hybridization, reverse transcription-polymerase chain reaction, and DNA sequencing. As expected from previous studies, numerous CD44 variants are expressed by actively proliferating and invaginating epithelia at sites of epithelial-mesenchymal interaction, e.g., in the developing tooth, nose and hair follicle. Our results show that most prominent amongst these variants at these sites are CD44(v3-v10), CD44(v4-v10), and CD44(v6-v10), as was also found previously in the apical ectodermal ridge of the developing limb (Yu et al. [1996]). The common pattern of expression of these three particular variants in active epithelia implies that they play an important role in morphogenesis. Also very prominent in morphogenetically active epithelia is CD44s; hyaluronan is uniformly absent from these epithelia and often also from associated mesenchyme with which they interact, supporting the previously documented role of CD44s in endocytic removal of hyaluronan.

Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.