ORIGINAL ARTICLE Annexin A2 depletion delays EGFR endocytic trafficking via cofilin activation and enhances EGFR signaling and metastasis formation M de Graauw 1 , L Cao 2,5 , L Winkel 3,5 , MHAM van Miltenburg 4 , SE le De ´ ve ´ dec 1 , M Klop 1 , K Yan 2 , C Pont 1 , V-M Rogkoti 1 , A Tijsma 1 , A Chaudhuri 1 , R Lalai 1 , L Price 1 , F Verbeek 2 and B van de Water 1 Enhanced epidermal growth factor receptor (EGFR) activity has been strongly linked to breast cancer progression and mediators of EGFR endocytosis may well be involved. We developed a semi-automated high-content fluorescence microscopy-based EGFR endocytosis screen to identify proteins that mediate EGFR endocytosis in human HBL100 breast cancer cells. Knockdown of 172 individual endocytosis and actin-regulatory genes with small interfering RNAs led to the identification of 14 genes of which the contribution to EGFR endocytosis in breast cancer is until now poorly defined, including DNAJC6, GDI2, FGD6, HAX1, NECAP2 and AnxA2. We show that depletion of the actin and endocytosis regulatory protein annexin A2 (AnxA2) in a panel of four triple negative breast cancer (TNBC) cell lines affected EGFR endocytosis. Depletion of AnxA2 in the aggressive and highly metastatic MDA-MB-231 TNBC cell line resulted in the inhibition of EGFR transport beyond the early endosomes. This inhibition coincided with enhanced epidermal growth factor (EGF)-induced cell migration and downstream signaling via c-Jun N-terminal kinase (JNK) and Akt. Moreover, AnxA2 knockdown increased lung metastasis formation in mice. The effect of AnxA2 knockdown on EGFR endocytosis in MDA-MB-231 was related to dephosphorylation/activation of the actin-severing protein cofilin, as re-expression of an inactive S3E- cofilin mutant, but not an active S3A-cofilin mutant, re-established EGFR endocytosis to control levels. Together, our data provide evidence for AnxA2 as a mediator of EGFR endocytosis and signaling in breast cancer via regulation of cofilin activation. Oncogene (2014) 33, 2610–2619; doi:10.1038/onc.2013.219; published online 24 June 2013 Keywords: AnxA2; breast cancer; EGFR; endocytosis; metastasis INTRODUCTION The epidermal growth factor receptor (EGFR) is important for normal growth and function of breast tissue and, when inappropriately activated, one of the main proteins involved in the development and progression of breast cancer. 1 A strong correlation has been found between the presence of high levels of EGFR in breast tumors and the aggressive potential of the tumor. 2 When EGFR is activated by binding to EGF, a chain of events is triggered. The EGFR dimerizes, which stimulates its intrinsic intracellular protein-tyrosine kinase activity, resulting in autophosphorylation of several tyrosine residues in the C-terminal domain of EGFR. Subsequently, different downstream signaling cascades are activated, including the Mitogen-activated protein kinase, JNK and Akt pathway. To prevent excessive growth and, thereby, formation of breast cancer, EGFR-dependent growth is, among others, controlled by endocytosis. 3 EGFR endocytosis is a multistep process, including receptor internalization at the plasma membrane, sorting in early endo- somes, transport to late endosomes, uptake in multi-vesicular bodies and degradation in the lysosomes. 4 As EGFR remains active within the endosomes until it is degraded, these endosomes can themselves form signaling platforms, resulting in propagation of intracellular signaling or in initiation of protein signaling cascades. 5 Over the past decades, many proteins have been shown to contribute to EGFR endocytosis, of which the adaptor protein growth factor receptor-bound Grb2 and the ubiquitin ligase Cbl have been described as two of the main regulators of EGFR endocytosis. 6,7 It has long been known that overexpression, mutations, deletions and production of autocrine ligands contribute to aberrant activation of EGFR in breast cancer. Yet, increasing evidence shows that aberrant EGFR signaling in breast cancer cells may also be caused by defects in the receptor endocytosis and degradation route. 8 A defective endocytosis route may result in persistent localization and activation of EGFR at the plasma membrane of cancer cells, as internalization of the receptor into early endosomes may be blocked or inhibited. 5 In addition, EGFR that is taken up into early endosomes may continue to signal from these endosomes, as passage to late endosomes and to degradation lysosomes may not take place. 5 Together, this results in enhanced EGFR signaling promoting cancer cell proliferation, invasion and metastasis formation. A better mechanistic understanding of the proteins that control EGFR endocytosis and thereby signaling in breast cancer cells may shed light on the mechanisms that control EGFR endocytosis and signaling in breast cancer. To identify regulators of EGFR endocytosis in breast cancer cells, we developed a semi-automated high-content fluorescent 1 Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands; 2 Imaging and Bio-informatics group, LIACS, Leiden University, Leiden, The Netherlands; 3 Biomechanics Laboratory, Erasmus MC, Rotterdam, The Netherlands and 4 Department of Molecular Pathology, Netherland Cancer Institute, Amsterdam, The Netherlands. Correspondence: Dr B van de Water or Dr M de Graauw, Division of Toxicology, LACDR, Leiden University, Einsteinweg 55, PO Box 9502, 2300 RA, Leiden, The Netherlands. E-mail: b.water@lacdr.leidenuniv.nl or m.de.graauw@lacdr.leidenuniv.nl 5 These authors contributed equally to this work. Received 18 April 2013; revised 19 April 2013; accepted 14 May 2013; published online 24 June 2013 Oncogene (2014) 33, 2610–2619 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc