Neuroblastoma, a tumor of the embryonic neural crest, is highly malignant and poorly responsive to conventional therapy, yet tumor cells retain the capacity for differentiation. This cancer is ultimately fatal in most patients and, thus, there is an urgent need to identify new agents and treatment strategies. Our long term goal is to understand the relationship between malignancy and cell differentiation state. Cell phenotype and amplification of the N-myc proto-oncogene are two factors that influence the malignant potential of neuroblastoma. In cell lines, N-myc expression and malignancy are dramatically affected by cell phenotype. Neuroblastic cells express high levels of N-myc and are tumorigenic whereas non-neuronal S-type cells from the same parental cell line contain markedly lower amounts of N-myc RNA/protein and are non-tumorigenic. The two cell types arise from a third cell type, malignant I-type stem cell. Moreover, experimentally induced changes in cell phenotype can directly affect N-myc expression and this oncoprotein, in turn, regulates specific downstream genes affecting cell growth and malignancy. These observations form the basis for our central hypothesis: Cell differentiation state, in particular the malignant stem cell phenotype, as well as N-myc expression levels directly regulate the malignant potential of neuroblastoma cells. One lineage-specific factor regulating N-myc expression is HuD, a neuronal-specific RNA-binding protein. HuD regulates the splicing/stability of N-myc mRNA and, in transfection experiments, it influences the amounts of cellular N-myc mRNA and protein. N-myc, in turn, regulates nestin and this neuroectodermal intermediate filament protein appears to mediate several of the oncogenic function of N-myc. The two aims of this proposal examine different aspects of our hypothesis. The focus of Aim #1 is to determine the mechanisms by which HuD interacts with N-nyc mRNA and thereby regulates its expression and, as a corollary, whether loss of one HuD allele is a critical factor in the development of N-myc amplification and tumor aggressiveness. Second, we will study the mechanism by which N-myc regulates nestin and how nestin augments the cell's malignant potential. Studies proposed in Aim # 2 will 1) establish whether the relative number of I-type stem cells in tumors correlates with tumor stage, aggressiveness, or event-free survival and is predictive for progressive disease and 2) isolate and characterize component phenotypes from tumors. Knowledge gained of the differentiation status of cells in tumors with a favorable or unfavorable prognosis and of factors that can directly affect cellular differentiation, and thereby malignant potential, is highly likely to have important application to the treatment of neuroblastoma.
