Abstract
BackgroundInteraction 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.MethodsCD73 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.ResultsStimulated 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.ConclusionThis study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.
Highlights
Interaction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer is important in the invasion and proliferation of cancer cells
Identification of molecules which form a complex with emmprin by Mass spectrometry (MS) analysis Proteins extracted from co-culture of tumor cells and fibroblasts, that had been cross-linked with BS3, were immunoprecipitated using anti-emmprin antibody and subjected to immunoblotting
In this study, we have for the first time reported that CD73 forms a complex with emmprin and regulates the production of Matrix metalloproteinase (MMP)-2 from fibroblasts, there has been a previous report providing evidence that suppression of CD73 leads to down-regulation of matrix metalloprotease-2 (MMP-2) [26]
Summary
Interaction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. The first extracellular Ig domain of emmprin is required for inducing MMPs production from fibroblasts; N-glycosylation is essential for activity [1, 3, 11, 12]. We have previously reported that MMP-2 production from fibroblasts is induced by emmprin’s first Ig domain (ECI) peptide, when substituted with chitobiose (the disaccharide with which N-glycosylation starts) [13]. We reported that synthetic peptides carrying a partial ECI construct of emmprin sequence without chitobiose could inhibit emmprin activity. The second peptide (emp#2), which contains a putative N-glycosylation site sequence, inhibited emmprinstimulated production of MMP-2 in co-cultures of fibroblasts [16]. Our findings suggested the formation of a complex with homophilic or heterophilic cis- or transinteractions between emmprin molecules within the plasma membrane or between emmprin and an unidentified cell surface molecule(s)
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