ICAM-2 Expression Mediates a Membrane-Actin Link, Confers a Nonmetastatic Phenotype and Reflects Favorable Tumor Stage or Histology in Neuroblastoma Karina Jin Yoon1*, Doris A. Phelps1, Rebecca A. Bush1, Joanna S. Remack1, Catherine A. Billups2, Joseph D. Khoury3 1 Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 2 Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 3 Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America Abstract The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/a- actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/a-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets. Citation: Yoon KJ, Phelps DA, Bush RA, Remack JS, Billups CA, et al. (2008) ICAM-2 Expression Mediates a Membrane-Actin Link, Confers a Nonmetastatic Phenotype and Reflects Favorable Tumor Stage or Histology in Neuroblastoma. PLoS ONE 3(11): e3629. doi:10.1371/journal.pone.0003629 Editor: Nils Cordes, Dresden University of Technology, Germany Received May 6, 2008; Accepted October 13, 2008; Published November 3, 2008 Copyright: 2008 Yoon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by CA21765 and American Lebanese Syrian Associated Charities. Competing Interests: The authors have declared that no competing interests exist. * E-mail: karina.yoon@stjude.org Introduction The actin network is a primary determinant of the motility of cells and organelles. This complex network regulates the movement of amoebae, distribution of bacteria within cells, cell migration in embryogenesis and angiogenesis, and the migratory and invasive (metastatic) potential of tumor cells. In tumor cells, motility and metastasis are thought to be regulated at least in part by the interaction of membrane-bound proteins with the actin cytoskeletal network [1–5]. Multiple signaling pathways and protein interactions modulate the motility and migration of tumor cells [3–8], but specific molecular interactions that control the complicated process of metastasis have been difficult to identify. In normal cells, the interaction of a particular membrane-bound cell adhesion molecule with a specific cytoplasmic linker protein has been associated with a unique cell function. In inactive neutrophils, for example, membrane-bound b2-integrin binds to the cytoplasmic linker protein talin. In contrast, in activated neutrophils b2-integrin binds to an alternate linker protein, a- actinin [9]. While both talin and a-actinin can bind directly to actin, it is not yet known how the association of b2-integrin with each linker proteins affects their binding to actin. In tumor cells, members of the ezrin-radixin-moesin (ERM) family of cytoplasmic actin cytoskeletal linker proteins play critical roles in the regulation of metastatic potential. ERM linker proteins, as well as a-actinin, merlin, and band 4.1 linker proteins [4–6,10– 12], have been implicated in tumor progression or in suppression of the metastatic potential of tumor cells. Cell adhesion molecules (CAMs) to which these linker proteins bind, such as deleted in colon cancer (DCC) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), are members of the IgG superfamily to which ICAM-2 belongs. DCC and CEACAM1 contribute to the adhesive, migratory, or invasive properties of several types of solid tumor cells [13–16] including neuroblastoma [17], but the molecular interactions that mediate these functions have been difficult to identify. The data herein suggest that in neuroblastoma cells the association of ICAM-2 with a-actinin and actin may represent such an interaction. Other functions have also been reported for ICAM-2. Particularly well characterized are its roles as a co-stimulatory signal for T cell stimulation [18] and in mediating neutrophil adhesion and migration through endothelial cell monolayers [19– 21]. These roles have been documented by in vivo [22–24] as well as in vitro methods. In specific cell types ICAM-2 also activates the PLoS ONE | www.plosone.org 1 November 2008 | Volume 3 | Issue 11 | e3629