title: N-glycosylation of ICAM-2 is required for ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma cells.
abstract: BACKGROUND
  Cell adhesion molecules (CAMs) are expressed ubiquitously. Each of the four families of CAMs is comprised of glycosylated, membrane-bound proteins that participate in multiple cellular processes including cell-cell communication, cell motility, inside-out and outside-in signaling, tumorigenesis, angiogenesis and metastasis. Intercellular adhesion molecule-2 (ICAM-2), a member of the immunoglobulin superfamily of CAMs, has six N-linked glycosylation sites at amino acids (asparagines) 47, 82, 105, 153, 178 and 187. Recently, we demonstrated a previously unknown function for ICAM-2 in tumor cells. We showed that ICAM-2 suppressed neuroblastoma cell motility and growth in soft agar, and induced a juxtamembrane distribution of F-actin in vitro. We also showed that ICAM-2 completely suppressed development of disseminated tumors in vivo in a murine model of metastatic NB. These effects of ICAM-2 on NB cell phenotype in vitro and in vivo depended on the interaction of ICAM-2 with the cytoskeletal linker protein α-actinin. Interestingly, ICAM-2 did not suppress subcutaneous growth of tumors in mice, suggesting that ICAM-2 affects the metastatic but not the tumorigenic potential of NB cells. The goal of the study presented here was to determine if the glycosylation status of ICAM-2 influenced its function in neuroblastoma cells.
  
  
  METHODS
  Because it is well documented that glycosylation facilitates essential steps in tumor progression and metastasis, we investigated whether the glycosylation status of ICAM-2 affected the phenotype of NB cells. We used site-directed mutagenesis to express hypo- or non-glycosylated variants of ICAM-2, by substituting alanine for asparagine at glycosylation sites, and compared the impact of each variant on NB cell motility, anchorage-independent growth, interaction with intracellular proteins, effect on F-actin distribution and metastatic potential in vivo.
  
  
  RESULTS
  The in vitro and in vivo phenotypes of cells expressing glycosylation site variants differed from cells expressing fully-glycosylated ICAM-2 or no ICAM-2. Most striking was the finding that mice injected intravenously with NB cells expressing glycosylation site variants survived longer (P ≤ 0.002) than mice receiving SK-N-AS cells with undetectable ICAM-2. However, unlike fully-glycosylated ICAM-2, glycosylation site variants did not completely suppress disseminated tumor development.
  
  
  CONCLUSIONS
  Reduced glycosylation of ICAM-2 significantly attenuated, but did not abolish, its ability to suppress metastatic properties of NB cells.
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Feduska
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Garcia
authors.forename: Stephanie B
authors.initials: SB
authors.lastname: Brennan
authors.forename: Su
authors.initials: S
authors.lastname: Bu
authors.forename: Leona N
authors.initials: LN
authors.lastname: Council
authors.forename: Karina J
authors.initials: KJ
authors.lastname: Yoon
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009361:Neoplasm Invasiveness / Q000473:pathology; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology*; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D000199:Actins; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; C480027:ICAM-2 protein, mouse