Bryan P. Toole

Hyaluronan-cell interactions in cancer and vascular disease.

Other articles in this series concentrate on normal physiological and cellular functions of hyaluronan. In this article we discuss the influences of hyaluronan on disease progression. Alterations in hyaluronan metabolism, distribution, and function have been documented in many diseases, e.g. arthritis, immune and inflammatory disorders, pulmonary and vascular diseases, and cancer (see Refs. 1 and 2). In this article we will concentrate on cancer and vascular disease because our knowledge in these areas has advanced rapidly over the past several years and because work in these areas has highlighted the importance of hyaluronan-cell interactions in cell behavior.

Glycosaminoglycans in Morphogenesis

Formation of a precisely organized, functional tissue or organ is the culmination of a complex series of specific cellular events, usually termed morphogenesis. These events involve several common types of cell behavior, notably movement, proliferation, shape change, recognition, and adhesion. At each stage of morphogenesis of an organ or tissue, the macromolecules present in the extracellular matrix or associated with the external cell surface are important in providing structural support for and environmental signals to the cells involved. These contributions in turn exert a considerable influence on the course of morphogenesis.

Proteoglycans and Hyaluronan in Morphogenesis and Differentiation

The objective of this chapter is to discuss the roles of hyaluronan (HA) and proteoglycans (PGs) in developmental processes and tissue remodeling. In the first section I have summarized recent progress on the molecular basis of HA and PG interactions with the surface of cells, since this is the most likely means whereby HA and PGs would influence cell behavior during development. This is followed by a review of cellular studies, mainly performed in vitro, that implicate HA and PGs in specific aspects of cell behavior that would be relevant to development in vivo. Finally, I have chosen three developmental systems to illustrate ways in which HA or PGs are involved in actual morphogenetic and differentiative processes.

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-CD44 Interactions in Cancer: Paradoxes and Possibilities

Hyaluronan is a prominent component of the micro-environment in most malignant tumors and can be prognostic for tumor progression. Extensive experimental evidence in animal models implicates hyaluronan interactions in tumor growth and metastasis, but it is also evident that a balance of synthesis and turnover by hyaluronidases is critical. CD44, a major hyaluronan receptor, is commonly but not uniformly associated with malignancy, and is frequently used as a marker for cancer stem cells in human carcinomas. Multivalent interactions of hyaluronan with CD44 collaborate in driving numerous tumor-promoting signaling pathways and transporter activities. It is widely accepted that hyaluronan-CD44 interactions are crucial in both malignancy and resistance to therapy, but major challenges for future research in the field are the mechanism of activation of hyaluronan-CD44 signaling in cancer cells, the relative importance of variant forms of CD44 and other hyaluronan receptors, e.g., Rhamm, in different tumor contexts, and the role of stromal versus tumor cell production and turnover of hyaluronan. Despite these caveats, it is clear that hyaluronan-CD44 interactions are an important target for translation into the clinic. Among the approaches that show promise are antibodies and vaccines to specific variants of CD44 that are uniquely expressed at critical stages of progression of a particular cancer, hyaluronidase-mediated reduction of barriers to drug access, and small hyaluronan oligosaccharides that attenuate constitutive hyaluronan-receptor signaling and enhance chemosensitivity. In addition, hyaluronan is being used to tag drugs and delivery vehicles for targeting of anticancer agents to CD44-expressing tumor cells. (Clin Cancer Res 2009;15(24):7462–8)

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. Int. J. Cancer 77:396–401, 1998.

Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells

Hyaluronan not only is an important structural component of extracellular matrices but also interacts instructively with cells during embryonic development, healing processes, inflammation, and cancer. It binds to several different types of cell surface receptors, including CD44, thus leading to co-regulation of important signaling pathways, notably those induced by activation of receptor tyrosine kinases. Consequently, interactions of both stromal and tumor cell-derived hyaluronan with tumor cells play important cooperative roles in several aspects of malignancy. This review focuses on cell autonomous hyaluronan-tumor cell interactions that lead to activation of receptor tyrosine kinases and enhanced drug resistance. Particular emphasis is placed on the role of hyaluronan-CD44 interactions in drug transporter expression and activity, especially in cancer stem-like cells that are highly malignant and resistant to chemotherapy. Antagonists of hyaluronan-CD44 interaction, especially small hyaluronan oligomers, may be useful in therapeutic strategies aimed at preventing tumor recurrence from these therapy-resistant sub-populations within malignant cancers.

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.

EMMPRIN-mediated MMP regulation in tumor and endothelial cells

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels.

CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A

Cyclophilin A (CyPA) is specifically incorporated into the virions of HIV-1 and has been shown to enhance significantly an early step of cellular HIV-1 infection. Our preliminary studies implicated CD147 as a receptor for extracellular CyPA. Here, we demonstrate a role for CyPA–CD147 interaction during the early steps of HIV-1 infection. Expression of human CD147 increased infection by HIV-1 under one-cycle conditions. However, susceptibility to infection by viruses lacking CyPA (simian immunodeficiency virus or HIV-1 produced in the presence of cyclosporin A) was unaffected by CD147. Virus-associated CyPA coimmunoprecipitated with CD147 from infected cells. Antibody to CD147 inhibited HIV-1 entry as evidenced by the delay in translocation of the HIV-1 core proteins from the membrane and inhibition of viral reverse transcription. Viruses whose replication did not require CyPA (SIV or mutant HIV-1) were resistant to the inhibitory effect of anti-CD147 antibody. These results suggest that HIV-1 entry depends on an interaction between virus-associated CyPA and CD147 on a target cell.