Delineating the biological importance and clinical importance of CyclinD1 in neuroblastoma

In most human cancers genetic lesions are present which disable key regulators of cell cycle progression. Frequently lesions occur in the p16 - CyclinD-CKK4 - Rb pathway. However, in the childhood tumour neuroblastoma no consistent cell cycle checkpoints abnormalities have yet been identified. Neuroblastoma is derived from sympathetic nervous precursor cells. The clinical course varies from spontaneous regression without any intervention to swift and fatal disease progression despite very aggressive therapy. In the neuroblastoma research group at the AMC we aim to delineate the molecular defects in neuroblastoma, underlying the wide spectrum of clinical behaviour. High throughput mRNA expression profiling by SAGE (Serial Analysis of Gene Expression) revealed that CyclinD1 was extremely high expressed in 5 out of 8 SAGE librarie3s of neuroblastoma cell lines and tumour. In none of the other 154 tissue's of which compiled SAGE libraries are available in the 'Human Transcriptome Map', higher expression levels were seen. Northern blot analysis of CyclinD1 expression showed overexpression in 68% of the 38 tested neuroblastoma cell line and tumour samples. Western blot analysis showed high CyclinD1 protein levels in those samples together with hyperphosphorylated Rb. At the DNA level we found 5 amplifications and one rearrangement in the 3'UTR of the CyclinD1 gnen in >200 tested tumour samples. Taken together our data support an important role for deregulated CyclinD1 expression in neuroblastoma tumorigenesis. In the majority of cases the mechanism is so far unidentified. In the proposed project we aim to answer the following questions: 1. Which upstream events cause the CyclinD1 overexpression in neuroblastoma? 2. What is the functional importance of CyclinD1 overexpression in neuroblastoma? 3. What is the clinical signinficance of CyclinD1 overexpression? For Q1 we will use promoter construct reporter assays ans EMSA to identify the direct transcriptional activators of CyclinD1 in neuroblastroma cell lines with up regulated CyclinD1 expression. We will also search for candidate upstream regulators by screening SAGE libraries of neuroblastoma, containing > 300.000 mRNA transcripts, for genes with patterns comparable to CyclinD1. These genes will be analysed in a large cell line and tumour panel by Nothern and Western blot. Candidate CyclinD1 regulaors will be further tested by transfection assays or expression interference analyses. For Q2 we will use RNAi to knock down the CyclinD1 protein levels and small molecue CDK inhibitors to inactivate the CyclinD1-CDK4 kinase function in several neuroblatoma cell lines. As readouts we will perform cell cycle distribution analyses, differential phophorylation analyses of the Rb protein on Western blot and growth kinetics measurements. For Q3 we will use immunohistochemical CyclinD1 detection on paraffin sections on our series of >200 neuroblastoma patients. We will analyse for correlations with clinical and genetic characteristics and perform uni and multivariate prognostic analysis. We expect that this project might yield new regulators of CyclinD1 and will establish the biological and clinical relevance of CyclinD1 overexpression in neuroblastoma. This might lead to new prognostic markers. The interference studies might lead to the formal in vitro validation of CyclinD1-CDK4 as a potential drug target in neuroblastoma.