Research Article Cyclin D1 and CDK4 Activity Contribute to the Undifferentiated Phenotype in Neuroblastoma Jan J. Molenaar,1 Marli E. Ebus,1 Jan Koster,1 Peter van Sluis,1 Carel J.M. van Noesel,3 Rogier Versteeg,1 and Huib N. Caron2 1Departments of Human Genetics and 2Pediatric Oncology, Emma Kinderziekenhuis, and 3Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Abstract this (6–8). The molecular mechanism underlying the differentiation pattern and the differences between these neuroblastic tumor types Genomic aberrations of Cyclin D1 (CCND1), CDK4, and CDK6 have been subject of several studies. Growth signaling pathways in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this and specific neurotrophins and their receptors have been found to pediatric tumor. Here, we report that analysis of Affymetrix determine differentiation patterns in nonmalignant neuroblasts expression data of primary neuroblastic tumors shows an and influence the differentiation state of neuroblastoma (9–11). extensive overexpression of Cyclin D1, which correlates with Recently, some papers functionally link neuronal differentiation histologic subgroups. Immunohistochemical analysis showed to cell cycle regulation, which frequently involves the G1 cell cycle overexpression of Cyclin D1 in neuroblasts and low Cyclin D1 entry point (12–15). This is a tightly controlled process by the expression in all cell types in ganglioneuroma. This suggests D-type cyclins and their kinase partners CDK4 and CDK6. In the an involvement of G -regulating genes in neuronal differen- presence of cyclin D, these kinases phosphorylate the pRb protein, 1 which then releases from the E2F transcription factor. Subsequent tiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partners CDK4 transcription of key regulator genes allows further progression and CDK6 in several neuroblastoma cell lines. The Cyclin D1 of the cell cycle (16). In neuroblastic tumors, several oncogenetic and CDK4 knockdown resulted in pRb pathway inhibition as events causing changes in cell cycle regulation have been shown by an almost complete disappearance of CDK4/CDK6- described. Amplifications of CDK4 and one mutation in CDK6 specific pRb phosphorylation, reduction of E2F transcriptional that disrupts p16 binding have been identified (17–19). We have reported amplification of the Cyclin D1 gene in 5 of 203 activity, and a decrease of Cyclin A protein levels. Phenotype ¶ analysis showed a significant reduction in cell proliferation, neuroblastic tumors and a rearrangement in the 3 untranslated region of the Cyclin D1 gene in one tumor (20). The sporadic aG1-specific cell cycle arrest, and, moreover, an extensive neuronal differentiation. Affymetrix microarray profiling of genomic aberrations of CDK4, CDK6, and Cyclin D1, together with small interfering RNA–treated cells revealed a shift in the findings of the very high Cyclin D1 expression levels, suggested expression profile toward a neuronal phenotype. Several new that dysregulation of the G1 entry checkpoint is an important cell potential downstream players are identified. We conclude that cycle aberration in neuroblastoma. In this paper, a detailed analysis of Cyclin D1 overexpression by neuroblastoma functionally depend on overexpression of Affymetrix profiling and immunohistochemical analysis of neuro- G1-regulating genes to maintain their undifferentiated phe- notype. [Cancer Res 2008;68(8):2599–609] blastic tumors reveals that the overexpression is specific for malignant neuroblasts. We show that silencing of Cyclin D1 and its kinase partner CDK4 causes an inhibition of the cyclin D1-pRb Introduction pathway, a G1 cell cycle arrest and growth arrest. Moreover, we Neuroblastic tumors develop from progenitor cell types that show a clear differentiation toward a neuronal cell type by immu- originate from the neural crest. These tumors are classified nofluoresence and Affymetrix Microarray analysis after Cyclin D1 into neuroblastoma and the more differentiated counter parts, and CDK4 silencing. ganglioneuroblastoma and ganglioneuroma. This histopathologic classification is based on morphologic features of the tumor. Undifferentiated neuroblastoma at one end of the spectrum Materials and Methods contain mainly neuroblasts, whereas on the other end, ganglio- Patient samples. The neuroblastic tumor panel used for Affymetrix neuroma consist of ganglion cells, Schwann cells, and stroma cells microarray analysis contains 88 neuroblastoma samples, 11 ganglioneuro- (1, 2). This spectrum of neuroblastic tumors is reflected in the blastoma samples, and 11 ganglioneuroma samples. All samples were development of nonmalignant neuroblasts that differentiate along derived from primary tumors of untreated patients. Material was obtained neural crest cell lineages to neuronal ganglia and chromaffin during surgery and immediately frozen in liquid nitrogen. N-Myc cells (3–5). The Schwann cells in neuroblastic tumors are believed amplifications and 1p deletions were all determined using Southern blot to be normal cells invading the tumor and not to originate from the analysis of tumor material and lymphocytes of the same patient. malignant neuroblasts, although some researchers have disputed Cell lines. Cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 20 mmol/L L-glutamine, 10 units/mL penicillin, and A j 10 g/mL streptomycin. Cells were maintained at 37 C under 5% CO2.For primary references of these cell lines, see Cheng et al. (21). Requests for reprints: Jan J. Molenaar, Department of Human Genetics, AMC, M1- RNA isolation, Northern blotting, and Affymetrix microarray 132, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands. Phone: 31-20-5667536; analysis. Total RNA of neuroblastoma tumors was extracted using Trizol Fax: 31-20-6918626; E-mail: [email protected]. I2008 American Association for Cancer Research. reagent (Invitrogen) according to the manufacturer’s protocol. RNA doi:10.1158/0008-5472.CAN-07-5032 concentration was determined using the NanoDrop ND-1000, and quality www.aacrjournals.org 2599 Cancer Res 2008; 68: (8). April 15, 2008 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research Cancer Res 2008; 68: (8). April 15, 2008 2600 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Neuroblastoma Depend on Cyclin D1 and CDK4 was determined using the RNA 6000 Nano assay on the Agilent 2100 and 671 to 691 (CD1-C) according to Genbank accession NM_053056. The Bioanalyzer (Agilent Technologies). For Northern blotting, 15 Ag of RNA CDK4 siRNA is targeting nucleotides 1062 to 1082 according to Genbank were electrophoresed through a 1% agarose gel containing 6.7% formalde- accession NM_000075. The CDK6 siRNA is targeting nucleotides 1112 to hyde and blotted on Hybond N membrane (Amersham) in 16.9 Â SSC and 1132 according to Genbank accession NM_001259. A previously designed 5.7% formaldehyde. The Cyclin D1 probe was generated by reverse siRNA directed against green fluorescent protein (GFP) was used as transcription–PCR. We used the following primers: 5¶-tcattgaacact- negative control (sense sequence: GACCCGCGCCGAGGUGAAGTT). Neuro- tcctctcc-3¶ and 5¶-gtcacacttgatcactctgg-3¶. Probes were sequence verified. blastoma cell lines were cultured for 24 h in 6-cm plates and transfected Probes were 32P labeled by random priming. We hybridized Northern blot with 5.5 Ag siRNA using Lipofectamine according to manufacturers’ filters for 16 h at 65jC in 0.5 mol/L Na2HPO4, 7% SDS, 1 mmol/L EDTA, and protocol. 50 Ag/mL herring sperm DNA. Measurement of signal intensity was Transactivation assays. The following luciferase constructs were used performed with a Fuji phosphor imager and Aida 2.41 software. Affymetrix in the transactivation assays: pGL3 TATAbasic-6xE2F (pGL3 containing a microarray analysis, fragmentation of RNA, labeling, hybridization to HG- TATA box and six E2F binding sites was previous tested for E2F selectivity U133 Plus 2.0 microarrays, and scanning were carried out according to the and was a kind gift of Prof. R. Bernards, Dutch Cancer Institute; ref. 22), manufacturer’s protocol (Affymetrix, Inc.). The expression data were Renilla luciferase vector under cytomegalovirus promoter (pRL-CMV). Cells normalized with the MAS5.0 algorithm within the GCOS program of were cultured for 24 h in six-well plates, and transfections were conducted Affymetrix. Target intensity was set to 100 (a1 = 0.04 and a2 = 0.06). If more using Lipofectamine 2000 according to manufacturers’ protocol. pGL3TA- then one probe set was available for one gene, the probe set with the highest TAbasic-6xE2F (0.8 Ag) vector was transfected, together with the 0.8 Ag expression was selected, considered that the probe set was correctly located pRL-CMV vector and Cyclin D1 siRNA or GFP siRNA. Dual-luciferase assays on the gene of interest. Public available Affymetrix expression data was were performed after 48 h using the Promega dual-luciferase reporter assay taken from the National Cancer Institute (NCI) Gene Expression Omnibus system. For each assay, three separate experiments were performed. database.4 Western blotting. The neuroblastoma cell lines were harvested on ice Immunohistochemistry. Paraffin-embedded tumors were cut into 4-Am and washed