Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.Oncogene (2012) 31, 5193 --5200; doi:10.1038/onc.2012.12; published online 30 January 2012Keywords: neuroblastoma; anaplastic lymphoma kinase; ALK; MYCN; transcription factor INTRODUCTION Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and accounts for B15% of all deaths due to paediatric tumours. 1,2 Genetically, many cases of neuroblastoma show amplification of the MYCN gene (B24% of all cases), deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. 1,3 --6 Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase (RTK) is mutated in both familial and somatic neuroblastoma. 7 --11 Moreover, these reports also indicated that downregulation or inhibition of ALK led to a marked decrease of cell proliferation, suggesting ALK as a critical factor in the initiation and progression of neuroblastoma. The ALK RTK was first described in the mid-nineties and aberrant ALK protein activity is now implicated in a range of nonhematopoietic, hematopoietic as well as neuroendocrine tumours (for a review see Palmer et al. 12 ). A recent meta-analysis of neuroblastoma has reported ALK gain-of-function mutations to be present at a frequency of 6.9% of investigated neuroblastoma tumours. Further, a comparison of the ALK mutation frequency in relation to genomic subtype revealed that ALK mutations were most frequently obs...
In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.
BackgroundEpigenetic mechanisms such as DNA methylation and histone modifications are important regulators of gene expression and are frequently involved in silencing tumor suppressor genes.MethodsIn order to identify genes that are epigenetically regulated in neuroblastoma tumors, we treated four neuroblastoma cell lines with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) either separately or in conjunction with the histone deacetylase inhibitor trichostatin A (TSA). Expression was analyzed using whole-genome expression arrays to identify genes activated by the treatment. These data were then combined with data from genome-wide DNA methylation arrays to identify candidate genes silenced in neuroblastoma due to DNA methylation.ResultsWe present eight genes (KRT19, PRKCDBP, SCNN1A, POU2F2, TGFBI, COL1A2, DHRS3 and DUSP23) that are methylated in neuroblastoma, most of them not previously reported as such, some of which also distinguish between biological subsets of neuroblastoma tumors. Differential methylation was observed for the genes SCNN1A (p < 0.001), PRKCDBP (p < 0.001) and KRT19 (p < 0.01). Among these, the mRNA expression of KRT19 and PRKCDBP was significantly lower in patients that have died from the disease compared with patients with no evidence of disease (fold change -8.3, p = 0.01 for KRT19 and fold change -2.4, p = 0.04 for PRKCDBP).ConclusionsIn our study, a low methylation frequency of SCNN1A, PRKCDBP and KRT19 is significantly associated with favorable outcome in neuroblastoma. It is likely that analysis of specific DNA methylation will be one of several methods in future patient therapy stratification protocols for treatment of childhood neuroblastomas.
BackgroundHypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma.ResultsIn the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material.In conclusionThis study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.
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