title: Annexin A2 depletion delays EGFR endocytic trafficking via cofilin activation and enhances EGFR signaling and metastasis formation.
abstract: 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.
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: de Graauw
authors.affiliation: Imaging and Bio-informatics group, LIACS, Leiden University, Leiden, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Cao
authors.affiliation: Biomechanics Laboratory, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Winkel
authors.affiliation: Department of Molecular Pathology, Netherland Cancer Institute, Amsterdam, The Netherlands.
authors.forename: M H A M
authors.initials: MH
authors.lastname: van Miltenburg
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: S E
authors.initials: SE
authors.lastname: le Dévédec
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Klop
authors.affiliation: Imaging and Bio-informatics group, LIACS, Leiden University, Leiden, The Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Yan
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: Pont
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: V-M
authors.initials: VM
authors.lastname: Rogkoti
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Tijsma
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Chaudhuri
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Lalai
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Price
authors.affiliation: Imaging and Bio-informatics group, LIACS, Leiden University, Leiden, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Verbeek
authors.affiliation: Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands.
authors.forename: B
authors.initials: B
authors.lastname: van de Water
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000818:Animals; D017306:Annexin A2 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D009362:Neoplasm Metastasis; D034622:RNA Interference; D015398:Signal Transduction
chemical_list: C472822:ANXA2 protein, human; D051339:Actin Depolymerizing Factors; D017306:Annexin A2; D066246:ErbB Receptors