Abstract
Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.
Highlights
IntroductionThe prognosis of patients with disseminated neuroblastoma is grim, with a 5-year survival rate of approximately 30%
Neuroblastoma is the most common extracranial solid tumor in childhood [14]
To identify the secreted proteins, a proteomic analysis was performed on the secretome of N-Myc amplified (SK-N-BE2) and non-amplified (SH-SY5Y) neuroblastoma cells
Summary
The prognosis of patients with disseminated neuroblastoma is grim, with a 5-year survival rate of approximately 30%. Amplification of the oncogene N-Myc is detected in approximately 20% of patients, and >10 copies is strongly correlated with higher tumour aggression and resistance to treatment. While the degree of malignancy, prognosis and resistance to therapy is associated with the amplification of N-Myc, its expression levels correlate with tumour growth and invasiveness [16, 17]. N-Myc amplification has been associated with increased levels of vascular endothelial growth factor (VEGF) thereby promoting angiogenesis in neuroblastoma [19]. Very little is known about the soluble secreted proteins that could modulate the aggressive phenotype of N-Myc amplified neuroblastoma cells. Bioinformatics analyses of the secreted proteins were carried out to short list potential N-Myc targets with relevance to neuroblastoma pathogenesis
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