beta1-integrin, integrin-linked kinase, and caveolin-1: Roles in neuroblastoma tumor biology

Neuroblastoma (NBL) tumors may develop from aberrant regulation of neural crest cell differentiation. Since integrins are important in neural crest cell migration, abnormal integrin expression and signaling may be one mechanism that promotes the development and progression of NBL. The current study compares integrin expression in human NBL cell lines to known tumorigenic characteristics. SHEP cells are less tumorigenic than SH-SY5Y and IMR-32 cells and express more beta1 integrin. Pulse-chase and treatment with cycloheximide demonstrated that SHEP cells express higher levels of beta1 integrin due to differences in protein turnover. Attachment and migration assay data confirmed that decreased beta1 integrin levels hinder adhesion to the extracellular matrix and promote migration in tumorigenic NBL cells.; Integrin-linked kinase (ILK) binds to beta1 integrin and is present in NBL tumors and cell lines. Our results suggest that SHEP cells initiate compensation mechanisms in response to ILK overexpression to prevent further transformation. Overexpression of ILK usually leads to altered expression of cell cycle regulators, decreased cell adhesion and apoptosis, and in vitro phosphorylation of Akt. In contrast, overexpression of beta1 integrin initiates differentiation in SHEP cells, which leads to apoptosis. Expression of wild-type ILK (ILK-wt) in SHEP cells increases beta1 integrin expression by 20%, but proliferation and apoptosis levels remain constant. Kinase deficient ILK induces decreased expression of glial fibrillary acidic protein, suggesting that the kinase domain of ILK is required to maintain SHEP cells in a differentiated state.; In addition to binding to beta1 integrin, ILK contains a caveolin-1 binding domain. Caveolin-I expression is down-regulated in tumors. We examined whether ILK and caveolin-1 interact in SHEP cells. ILK is present in caveolin-enriched membranes and colocalizes with caveolin-1 in immunocytochemistry experiments. Immunoprecipitation with anti-caveolin-1 coimmunoprecipitates a 59 kD protein that reacts with an anti-ILK antibody, and this is reduced in cells expressing ILK with a mutant caveolin-1 binding domain (ILK-mutCavbd). Lastly, affinity chromatography with a biotinylated caveolin-scaffolding domain peptide affinity purifies ILK-wt but not ILK-mutCavbd. These data suggest that the caveolin-binding domain of ILK and the caveolin-scaffolding domain of caveolin-I mediate complex formation in SHEP cells.