Bryan P. Toole

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.

CD147 (basigin; EMMPRIN), hyaluronan, and hyaluronan receptors (e.g., CD44) are intimately involved in several phenomena that underlie malignancy. A major avenue whereby they influence tumor progression is most likely their role in the characteristics of cancer stem cells (CSCs), subpopulations of tumor cells that exhibit chemoresistance, invasiveness, and potent tumorigenicity. Both CD147 and hyaluronan have been strongly implicated in chemoresistance and invasiveness, and may be drivers of CSC characteristics, since current evidence indicates that both are involved in epithelial‐mesenchymal transition, a crucial process in the acquisition of CSC properties. Hyaluronan is a prominent constituent of the tumor microenvironment whose interactions with cell surface receptors influence several signaling pathways that lead to chemoresistance and invasiveness. CD147 is an integral plasma membrane glycoprotein of the Ig superfamily and cofactor in assembly and activity of monocarboxylate transporters (MCTs). CD147 stimulates hyaluronan synthesis and interaction of hyaluronan with its receptors, in particular CD44 and LYVE‐1, which in turn result in activation of multiprotein complexes containing members of the membrane‐type matrix metalloproteinase, receptor tyrosine kinase, ABC drug transporter, or MCT families within lipid raft domains. Multivalent hyaluronan–receptor interactions are essential for formation or stabilization of these lipid raft complexes and for downstream signaling pathways or transporter activities. We conclude that stimulation of hyaluronan–receptor interactions by CD147 and the consequent activities of these complexes may be critical to the properties of CSCs and their role in malignancy. Anat Rec, 2019.

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent enzymes involved in various pathologic and physiologic processes. In cancer, MMPs contribute to processes from tumour initiation to establishment of distant metastases. Complex signalling and protein transport networks regulate MMP synthesis, cell surface presentation and release. Earlier attempts to disrupt MMP activity in patients have proven to be intolerable and with underwhelming clinical efficacy; thus targeting ancillary proteins that regulate MMP activity may be a useful therapeutic approach. Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally characterized as a factor present on lung cancer cells, which stimulated collagenase (MMP-1) production in fibroblasts. Subsequent studies demonstrated that EMMPRIN was identical with several other protein factors, including basigin (Bsg), all of which are now commonly termed CD147. CD147 modulates the synthesis and activity of soluble and membrane-bound [membrane-type MMPs (MT-MMPs)] in various contexts via homophilic/heterophilic cell interactions, vesicular shedding or cell-autonomous processes. CD147 also participates in inflammation, nutrient and drug transporter activity, microbial pathology and developmental processes. Despite the hundreds of manuscripts demonstrating CD147-mediated MMP regulation, the molecular underpinnings governing this process have not been fully elucidated. The present review summarizes our present knowledge of the complex regulatory systems influencing CD147 biology and provides a framework to understand how CD147 may influence MMP activity.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and KSHV activation of mitogen-activated protein kinases (MAPKs) initiates a number of key pathogenic determinants of KS. Direct inhibition of signal transduction as a therapeutic approach presents several challenges, and a better understanding of KSHV-induced mechanisms regulating MAPK activation may facilitate the development of new treatment or prevention strategies for KS. MAPK phosphatases, including dual-specificity phosphatase-1 (DUSP1), negatively regulate signal transduction and cytokine activation through MAPK dephosphorylation or interference with effector molecule binding to MAPKs, including the extracellular signal-regulated kinase (ERK). We found that ERK-dependent latent viral gene expression, the induction of promigratory factors, and cell invasiveness following de novo infection of primary human endothelial cells are in part dependent on KSHV suppression of DUSP1 expression during de novo infection. KSHV-encoded miR-K12-11 upregulates the expression of xCT (an amino acid transporter and KSHV fusion/entry receptor), and existing data indicate a role for xCT in the regulation of 14-3-3β, a transcriptional repressor of DUSP1. We found that miR-K12-11 induces endothelial cell secretion of promigratory factors and cell invasiveness through upregulation of xCT-dependent, 14-3-3β-mediated suppression of DUSP1. Finally, proof-of-principle experiments revealed that pharmacologic upregulation of DUSP1 inhibits the induction of promigratory factors and cell invasiveness during de novo KSHV infection. These data reveal an indirect role for miR-K12-11 in the regulation of DUSP1 and downstream pathogenesis.

A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia to breach these structural barriers. Invadopodia are actin-based, lipid-raft-enriched membrane protrusions containing membrane-type-1 matrix metalloproteinase (MT1-MMP; also known as matrix metalloproteinase 14; MMP14) and several signaling proteins. CD147 (emmprin, basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in many systems. In this study we show that upregulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodia-like structures and co-fractionate in membrane compartments with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, probably through assembly of MT1-MMP-containing complexes within lipid-raft domains of the invadopodia.

Background: Emmprin, a glycoprotein containing two Ig domains, is enriched on tumor cell surfaces and stimulates matrix metalloproteinase (MMP) production by adjacent stromal cells. Its first Ig domain (ECI) contains the biologically active site. The dependence of emmprin activity on N-glycosylation is controversial. We investigated whether synthetic ECI with the shortest sugar is functionally active. Methods: The whole ECI peptides carrying sugar chains, a chitobiose unit or N-linked core pentasaccharide, were synthesized by the thioester method and added to fibroblasts to examine whether they stimulate MMP-2 production. Results: ECI carrying a chitobiose unit, ECI-(GlcNAc) ₂, but not ECI without a chitobiose unit or the chitobiose unit alone, dose-dependently stimulated MMP-2 production by fibroblasts. ECI with longer chitobiose units, ECI-[(Man)₃(GlcNAc)₂], also stimulated MMP-2 production, but the extent of its stimulation was lower than that of ECI-(GlcNAc)₂. Conclusions: Our results indicate that ECI can mimic emmprin activity when substituted with chitobiose, the disaccharide with which N-glycosylation starts.

Purpose: CD44 is one of the most common markers used for identification of highly tumorigenic subpopulations of human carcinoma cells, but little is known about the function of CD44 or its major ligand, hyaluronan, in these cells. The purpose of this study was to investigate the involvement of hyaluronan and its interaction with CD44 in the properties of a tumorigenic subpopulation of primary ovarian carcinoma cells. Experimental Design: A tumorigenic subpopulation was identified in ascites fluids from ovarian carcinoma patients by expression of high CD133 levels. Treatment with small hyaluronan oligosaccharides, which dissociate constitutive hyaluronan polymer-CD44 interactions, was used to test the importance of hyaluronan-CD44 interaction in assembly of multidrug and monocarboxylate transporters and receptor tyrosine kinases in the plasma membrane of cells with high CD133 levels, and in the tumorigenic capacity of the CD133-high subpopulation. Results: Although total CD44 levels were similar in cells with high or low CD133 expression, CD44 was present in close association with transporters, receptor tyrosine kinases, and emmprin (CD147) in the plasma membrane of cells with high CD133 levels. Treatment with small hyaluronan oligosaccharides reduced association of the transporters and receptor tyrosine kinases with CD44 in the plasma membrane, diminished drug transporter activity, and inhibited i.p. tumorigenesis in these cells. Conclusions: We conclude that hyaluronan-CD44 interaction plays an important role in the properties of highly tumorigenic cells by stabilizing oncogenic complexes in their plasma membrane, and that treatment with hyaluronan-CD44 antagonists provides a logical therapeutic approach for abrogating the properties of these cells. (Clin Cancer Res 2009;15(24):7593–601)