Cyclin D1 and CDK4 Activity Contribute to the Undifferentiated Phenotype in Neuroblastoma

Abstract

Genomic aberrations of Cyclin D1 (CCND1), CDK4, and CDK6 in neuroblastoma indicate that dysregulation of the G 1 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 G 1 -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 G 1 -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 G 1 -regulating genes to maintain their undifferentiated phenotype. [Cancer Res 2008;68(8):2599-609]

Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo

“…We also showed that knockdown of CND1 reproduces part of the phenotype observed by inhibition of the NFkB signaling. In good agreement with our findings, CND1 knockdown in neuroblasts promoted a reduction in cell proliferation and an extensive neuronal differentiation (Molenaar et al, 2008). 1 and 2), and a scrambled shRNA-negative control (lane 3), along with an EGFP-containing vector.…”

“…CND1 has been suggested to maintain an undifferentiated phenotype of neuroblasts (Molenaar et al, 2008). As this gene was consistently upregulated in differentiated cells (Figure 1e), we evaluated its contribution to the NFkB-mediated control of differentiating GICs.…”

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

“…Upon mitogenic stimulation pRb functional inactivation is promoted through sequential phosphorylation by cyclin D-CDK4/6 and cyclin E-CDK2 complexes and results in the release of active E2F1-3 transcription factors capable of inducing the expression of genes encoding proteins crucial for the G1 to S phase transition [41,42]. In neuroblastoma cells cyclin D1 and CDK4 overexpression has been reported to play a role in pRb inactivation [30]. In this study, we show that in imatinib treated cells pRb phosphorylation at the CDK2-specific Thr-821 and CDK4-specific Ser-780 was decreased and in parallel the expression of two E2F responsive genes previously reported to be upregulated in neuroblastoma cells: SKP2 and cyclin A [30,31] was downregulated.…”

“…To evaluate the effects of imatinib on CDK4 activity, the phosphorylation status of pRb at the CDK4-specific Ser-780 [30] was investigated. As shown in Fig.…”

“…This led us to hypothesize that in imatinib treated cells E2F activity is decreased. To test this hypothesis, the expression of SKP2 and cyclin A, two E2F responsive genes known to be upregulated in neuroblastoma cells [30,31] was evaluated. As shown in Fig.…”