Abstract
BackgroundNeuroblastoma is the most common extracranial solid malignancy in childhood, responsible for 15% of all pediatric cancer deaths. It is an heterogeneous disease that does not always respond to classical therapy; so the identification of new and specific molecular targets to improve existing therapy is needed. We have previously demonstrated the involvement of the Notch pathway in the onset and progression of neuroblastoma. In this study we further investigated the role of Notch signaling and identified Delta-like 1 (DLL1) as a novel molecular target in neuroblastoma cells with a high degree of MYCN amplification, which is a major oncogenic driver in neuroblastoma. The possibility to act on DLL1 expression levels by using microRNAs (miRNAs) was assessed.MethodsDLL1 mRNA and protein expression levels were measured in three different neuroblastoma cell lines using quantitative real-time PCR and Western Blot analysis, respectively. Activation of the Notch pathway as a result of increased levels of DLL1 was analyzed by Immunofluorescence and Western Blot methods. In silico tools revealed the possibility to act on DLL1 expression levels with miRNAs, in particular with the miRNA-34 family. Neuroblastoma cells were transfected with miRNA-34 family members, and the effect of miRNAs transfection on DLL1 mRNA expression levels, on cell differentiation, proliferation and apoptosis was measured.ResultsIn this study, the DLL1 ligand was identified as the Notch pathway component highly expressed in neuroblastoma cells with MYCN amplification. In silico analysis demonstrated that DLL1 is one of the targets of miRNA-34 family members that maps on chromosome regions that are frequently deregulated or deleted in neuroblastoma. We studied the possibility to use miRNAs to target DLL1. Among all miRNA-34 family members, miRNA-34b is able to significantly downregulate DLL1 mRNA expression levels, to arrest cell proliferation and to induce neuronal differentiation in malignant neuroblastoma cells.ConclusionsTargeted therapies have emerged as new strategies for cancer treatment. This study identified the Notch ligand DLL1 as a novel and attractive molecular target in childhood neuroblastoma and its results could help to devise a targeted therapy using miRNAs.
Highlights
Neuroblastoma is the most common extracranial solid malignancy in childhood, responsible for 15% of all pediatric cancer deaths
The anti-oncogenic activity of miRNA-34a has been demonstrated in cancer cells of the lung [30, 31], pancreas [32, 33], brain [34, 35], ovary [36], prostate [37] as well as in lymphoma and leukemia [38]. miRNA-34a inhibits the propagation properties of tumor-initiating cells derived from medulloblastoma [39] and it is downregulated in glioblastoma tissues, where its overexpression could suppress cell proliferation and induce apoptosis, indicating that this miRNA may act as tumor suppressor in this type of tumor [40]. miRNA-34b is significantly downregulated in prostate cancer and its reconstitution induced anti-proliferative and antimigratory effects and suppressed tumor growth in an in vivo xenograft nude mouse model, suggesting the tumor suppressor function of this miRNA [41]
Delta-like 1 (DLL1) is the notch pathway component highly expressed in MYCN amplified neuroblastoma cells At first, the role of Notch pathway components was investigated in neuroblastoma cell lines with different MYCN gene amplification degree, which is related to aggressiveness and malignant phenotype
Summary
Neuroblastoma is the most common extracranial solid malignancy in childhood, responsible for 15% of all pediatric cancer deaths. In this study we further investigated the role of Notch signaling and identified Delta-like 1 (DLL1) as a novel molecular target in neuroblastoma cells with a high degree of MYCN amplification, which is a major oncogenic driver in neuroblastoma. Neuroblastoma is an embryonic tumor of the sympathetic nervous system which arises during fetal or early postnatal life from sympathetic cells derived from the neural crest [1] It is the most common solid extracranial malignancy of childhood and is responsible for 15% of all pediatric cancer deaths. This study identified Notch ligand DLL1 as a new and specific molecular target in childhood neuroblastoma, suggesting that miRNAs could be a novel therapeutic tool to develop an effective strategy to attack “DLL1 positive” neuroblastoma
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