Cyclin D1 and CDK4 activity contribute to the undifferentiated phenotype in neuroblastoma

Genomic aberrations of Cyclin D1 (CCND1), CDK4, and CDK6 in neuroblastoma indicate that dysregulation of the G₁ entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here, we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive overexpression of Cyclin D1, which correlates with histologic subgroups. Immunohistochemical analysis showed overexpression of Cyclin D1 in neuroblasts and low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G₁-regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partners CDK4 and CDK6 in several neuroblastoma cell lines. The Cyclin D1 and CDK4 knockdown resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4/CDK6-specific pRb phosphorylation, reduction of E2F transcriptional activity, and a decrease of Cyclin A protein levels. Phenotype analysis showed a significant reduction in cell proliferation, a G₁-specific cell cycle arrest, and, moreover, an extensive neuronal differentiation. Affymetrix microarray profiling of small interfering RNA-treated cells revealed a shift in expression profile toward a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on overexpression of G1-regulating genes to maintain their undifferentiated phenotype.