The Role of Nucleolin in B-cell Lymphomas and Fas-Mediated Apoptotic Signaling

The death receptor Fas has a key role in mediating homeostasis, elimination of defective cells and more recently implicated in cancer promotion. Many effective anti-cancer therapies depend on Fas-mediated apoptosis to eradicate tumor cells and ineffective Fas-apoptotic signaling is a basis for primary as well as acquired resistance to chemotherapy. We hypothesized that Fas is subjected to direct regulation by inhibitory proteins attained by cancer cells. To screen for potential binding modulators of Fas, we analyzed lymphoma cells for Fas binding proteins. This purification scheme identified high scoring peptides derived from nucleolin, a nuclear protein known to be overexpressed in cancer. We confirmed binding of nucleolin to Fas and the presence of nucleolin-Fas complexes on the surface of lymphoma cells. Using deletion mutants of nucleolin, we identified RBD 4 and glycine/arginine rich region of nucleolin to be required for the binding to Fas. BJAB cells, a Burkitts lymphoma cell line, with partial knockdown of nucleolin showed significantly higher rates of apoptosis in response to Fas agonists and increased ligand binding when compared to non-target controls. Transfection of mice with nucleolin-expressing plasmids showed significantly higher survival rates in nucleolin-transfected mice than vector control- and non-Fas-binding nucleolin mutant- transfected mice after lethal Fas agonist challenge. We next examined the expression of nucleolin in human B-cell lymphomas. We observed that nucleolin is overexpressed in multiple B-cell lymphomas and its localization changes in transformed cells. In a tissue microarray analysis we showed that although total nucleolin levels did not correlate with patient outcome, including progression free survival and overall survival, the nucleolin levels did correlate with proliferative staining. We developed a method for detecting nucleolin surface expression levels and showed that surface nucleolin expression is increased in aggressive B-cell lymphoma subtypes including MCL and DLBCL yet is low in healthy B cells and chronic lymphocytic leukemia, a low grade cancer. We also determined that nucleolin surface expression correlates with a worsening prognostic index. Results from this investigation provide the first evidence of nucleolin overexpression as a clinical correlate for worsening prognosis of lymphomas. The potential underlying mechanism involves blocking of Fas-mediated apoptotic death pathway through direct binding of nucleolin to the Fas receptor on the extracellular surface of cancer cells