The EphA3 Receptor Is Expressed in a Subset of Rhabdomyosarcoma Cell Lines and Suppresses Cell Adhesion and Migration Noretta Clifford, 1 Loraine M. Smith, 1 James Powell, 1 Stefan Gattenlo ¨ hner, 2 Alexander Marx, 2,3 and Rosemary O’Connor 1 * 1 Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, University College Cork, Cork, Ireland 2 Institute of Pathology, University of Wu ¨ rzburg, Wu ¨ rzburg, Germany 3 Institute of Pathology, University Hospital Mannheim, University of Heidelberg, Heidelberg, Germany ABSTRACT Elevated expression of the Eph receptor tyrosine kinase EphA3 is associated with lymphocytic leukaemia, but little is known about its expression or function in solid tumours. Out of a panel of cancer cell lines, we found that EphA3 was expressed only on two rhabdomyosarcoma (RMS) cell lines of the embryonal histological subtype and on one of the alveolar RMS subtype, whereas it was not detected on two other cell lines of the alveolar subtype. Other EphA receptors (1–7) were, either not expressed in any, or expressed in all five RMS cell lines. Stimulation of EphA3-expressing TE671 and RD RMS cells with ephrinA5 resulted in loss of adhesion to fibronectin, decreased migration towards the stromal cell-derived growth factor-I (SDF-I), increased EphA3 phosphorylation, and increased Rho GTPase activity. In contrast, ectopic expression of EphA3 in the EphA3 negative CRL2061 cell line resulted in decreased cell adhesion. Finally, suppression of EphA3 expression by siRNA in RD cells results in increased SDF-I-mediated motility. These data indicate that EphA3 expression may define subsets of RMS tumours, and that EphA3 suppresses motility through regulation of Rho GTPases in RMS cells. J. Cell. Biochem. 105: 1250– 1259, 2008. ß 2008 Wiley-Liss, Inc. KEY WORDS: EphA3; RHABDOMYOSARCOMA; MIGRATION W ith 16 members, the Eph receptor family is the largest subgroup of the receptor tyrosine kinase (RTK) family. It is divided into two subclasses, A and B, based on distinct structural properties of their ligands, the ephrins [Committee, 1997]. The Eph receptors and ephrins are membrane-bound proteins whose interactions initiate unique bidirectional signalling events in both the receptor- and the ligand-expressing cells [Dodelet and Pasquale, 2000]. Ephs and ephrins are frequently expressed on adjacent or even the same cell populations and exhibit a characteristic promiscuity, with the possibility for ephrin ligands to ligate and activate more than one member of the Eph receptor family [Gale et al., 1996; Smith et al., 2004a]. Crosstalk between Ephs and other receptors has also been reported as well as the formation of stable receptor complexes between different members of the Eph family, all of which adds to the complexity of Eph/ephrin bidirectional signalling [Murai and Pasquale, 2003]. Altered expression of Ephs and ephrins is associated with angiogenesis and tumour vasculature in many types of human cancers, including breast, lung, prostate cancers, melanoma, and leukaemia [Dodelet and Pasquale, 2000; Clevers and Batlle, 2006], reviewed in Surawska et al. [2004]. EphA2 expression is associated with aggressive cancer progression and metastasis [Easty et al., 1995; Zelinski et al., 2001]. The EphA3 receptor (also called HEK) is increased without apparent amplification or rearrangement in human lymphoid tumour cell lines, which raises the possibility that overexpression of EphA3 is a contributing factor in lymphoid malignancy [Wicks et al., 1992]. EphB4 was found to be highly expressed in the outer layer of the tumour cell mass in grade III Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 105:1250–1259 (2008) 1250 N. Clifford and L.M. Smith contributed equally to the study. Additional supporting information may be found in the online version of this article. Grant sponsor: Cancer Research Ireland; Grant sponsor: Higher Education Authority and Science Foundation Ireland. *Correspondence to: Rosemary O’Connor, Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, University College Cork, Cork, Ireland. E-mail: r.oconnor@ucc.ie Received 6 April 2008; Accepted 18 August 2008  DOI 10.1002/jcb.21926  2008 Wiley-Liss, Inc. Published online 22 September 2008 in Wiley InterScience (www.interscience.wiley.com).