Degradation of the surrounding extracellular matrix (ECM) by matrix metalloproteinases (MMPs) drives invasion and metastasis of cancer cells. We previously demonstrated that tetraspanin CD9 expression upregulates pro-MMP-9 expression and release and promotes cellular invasion in a human fibrosarcoma cell line (HT1080). These events were dependent upon the highly functional second extracellular loop of CD9. We report here that the epidermal growth factor receptor (EGFR) tyrosine kinase expression and activity are involved in the CD9-mediated increase in pro-MMP-9 release and cellular invasion. Pro-MMP-9 expression was significantly decreased in a dose-dependent manner using first a broad spectrum receptor tyrosine kinase inhibitor and multiple specific EGFR inhibitors in CD9-HT1080 cells. Furthermore, gefitinib treatment of CD9-HT1080 cells reduced invasion through matrigel. EGFR knockdown using short interfering RNA resulted in decreased pro-MMP-9 expression and release into the media and subsequent cellular invasion without affecting CD9 expression or localization. Conclusively, this study points to EGFR as a key mediator between CD9-mediated pro-MMP-9 release and cellular invasion of HT1080 cells. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.