N-glycosylation of ICAM-2 is required for ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma cells

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.

Reduced glycosylation of ICAM-2 significantly attenuated, but did not abolish, its ability to suppress metastatic properties of NB cells.

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.

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.