Abstract 275: The shedding of epidermal growth factor receptor is mediated by MMP-2 or MMP-9 and stimulated by fibronectin in MDA-MB-468 cells

Activation of the epidermal growth factor receptor (EGFR) is essential for cell proliferation and tissue homeostasis. Alterations in the ERBB signaling pathway have been detected in several human cancers. Soluble isoforms of EGFR generated by proteolytic cleavage of the membrane bound receptor have been identified in several cell lines. The shedding of EGFR in MDA-MB-468 cells generates a fragment of approximately 110 kDa that comprises the extracellular domain plus four amino acids of the transmembrane domain of the full-length receptor. The shedding of EGFR in MDA-MB-468 is activated by two ligands of the receptor (EGF, TGF-alpha), fetal bovine serum, an inhibitor of phosphatases (pervanadate) and by an activator of metalloproteases (i.e. APMA). However, the proteolytic activity that cleaves EGFR is unknown. Studies using metalloprotease inhibitors have suggested that matrix metalloproteases (MMPs) are involved in the shedding of EGFR. Here, we show that the shedding of EGFR increased in MDA-MB-468 cells treated with activated MMP-2 and MMP-9. Similarly, we observed cleavage of the receptor when EGFR (Sigma) or membrane preparations from MDA-MB-468 cells were incubated in vitro with activated MMP-2 or MMP-9. This cleavage was blocked when antibodies against MMP-2 or MMP-9 were included in the incubation mixture. Since fetal bovine serum increases the shedding of EGFR in MDA-MB-468 cells, we evaluated whether other serum components in addition to EGF and TGF-alpha have similar effect. Our results showed that the shedding of EGFR increased when fibronectin (Sigma) was added to MDA-MB-468 cells growing in serum free culture medium. This increase in the shedding of EGFR by fibronectin was dose and time dependent and was abolished when antibodies against α1β5 integrin was added to the incubation medium. Together, these results strongly suggest that the shedding of EGFR is mediated by MMP-2 and MMP-9 and therefore, it is of physiological relevance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 275.