Nestin is a potential mediator of malignancy in human neuroblastoma cells
Neuroblastoma is a peripheral nervous system cancer arising from the embryonic neural crest. Neuronal precursor cells of the neural crest normally express N-myc in a cell cycle- and differentiation-dependent manner, but this oncogene is amplified and overexpressed in a subset of neuroblastomas. Patients with N-myc amplified tumors exhibit rapid disease progression and a poor prognosis. Another protein whose elevated expression has been linked to aggressiveness in nervous system tumors is the intermediate filament nestin. Nestin is a neuroepithelial stem cell marker that has been shown to function in cell differentiation and in organizing the cytoskeleton. In this study, we have examined the role of nestin in human neuroblastoma cells. Northern and Western blot analyses of nestin expression in 22 clones or populations derived from 14 different tumors revealed that nestin is (1) is present in all neuroblastoma cell lines examined, (2) expressed at similar levels among neuroblastic (N-type), substrate-adherent (S-type), and intermediate (1-type) cell variants from any one cell line, and (3) expressed in a cell cycle-dependent manner. Interestingly, N-type cell lines that overexpress N-myc also have high levels of nestin. To determine whether these two proteins interact, I stably transfected LA1-55n N-type cells with a 265bp antisense sequence from the fourth exon of the nestin gene. Nestin protein levels are 2-fold lower in antisense-transfected clones. Concomitant with reduced nestin protein are an increase in population doubling time, a ∼5-fold decrease in colony formation in soft agar, and a marked decrease in cell motility. However, cell invasivity, phenotype, and Nmyc protein levels are unchanged. When the N-myc amplified cell line LA1-55n was transfected with an antisense N-myc construct, the endogenous levels of N-myc protein decreased, as did the levels of nestin protein. These antisense transfectants also displayed slower doubling times and decreased motility. Conversely, when the N-myc nonamplified cell line, SH-SY5Y, was transfected with a sense N-myc construct, the levels of N-myc protein increased, as did the levels of nestin protein. These sense transfectants displayed faster growth rates and increased motility as well. Finally, gel mobility shift assays show that N-myc binds specifically to E-boxes in the regulatory second intron of the nestin gene. Together, these studies reveal that nestin (1) is upregulated with N-myc overexpression, (2) influences malignant potential, (3) affects both migratory ability and growth rate of N-type human neuroblastoma cells and (4) is downstream of and regulated by N-myc. Thus, nestin may be one effector through which N-myc influences neuroblastoma metastasis and malignancy.
Thomas, Sharon K, "Nestin is a potential mediator of malignancy in human neuroblastoma cells" (2003). ETD Collection for Fordham University. AAI3083160.