Abstract
P53 mutations are responsible for drug-resistance of tumour cells which impacts on the efficacy of treatment. Alternative tumour suppressor pathways need to be explored to treat p53- deficient tumours. The E3 ubiquitin ligase, ITCH, negatively regulates the tumour suppressor protein TP73, providing a therapeutic target to enhance the sensitivity of the tumour cells to the treatment. In the present study, two p53-mutant neuroblastoma cell lines were used as in vitro models. Using immunostaining, western blot and qPCR methods, we firstly identified that ITCH was expressed on p53-mutant neuroblastoma cell lines. Transfection of these cell lines with ITCH siRNA could effectively silence the ITCH expression, and result in the stabilization of TP73 protein, which mediated the apoptosis of the neuroblastoma cells upon irradiation treatment. Finally, in vivo delivery of the ITCH siRNA using nanoparticles to the neuroblastoma xenograft mouse model showed around 15–20% ITCH silencing 48 hours after transfection. Our data suggest that ITCH could be silenced both in vitro and in vivo using nanoparticles, and silencing of ITCH sensitizes the tumour cells to irradiation treatment. This strategy could be further explored to combine the chemotherapy/radiotherapy treatment to enhance the therapeutic effects on p53-deficient neuroblastoma.
Highlights
Neuroblastoma is a childhood malignant tumour accounting for ~13% of all paediatric cancer mortality[1]
Anti-GD2 antibody therapies combined with granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2) and 13-cis-retinotic acid have improved the 2-year survival rate by 20%40,43,44, but neuroblastoma cells with low GD2 expression fail to respond to this therapy[45] and so the prognosis of high-risk neuroblastoma remains very poor[43,46] and new treatments are required
We have previously described nanoformulations for delivery of plasmid DNA encoding IL-2 and IL-1235,36 for therapy of murine form of neuroblastoma but here we explore a new approach in human p53-deficient neuroblastoma cells based on siRNA delivery to silence ITCH ligase and so induce p73 expression to mediate apoptosis in response to DNA damaging agents or radiation
Summary
Neuroblastoma is a childhood malignant tumour accounting for ~13% of all paediatric cancer mortality[1]. P73 mutations are infrequent in human cancers[17] including neuroblastomas[19,20], making it an attractive gene to manipulate for therapeutic intervention of the p53-null tumours. In addition to p73, ITCH regulates other tumour suppressor genes such as large tumour suppressor 1 (LATS1)[28,29], p6330,31, and RASSF5/ NORE132, providing an attractive therapeutic target for tumour therapy. We tested the hypothesis that modulation of ITCH expression would enhance the sensitivity of the tumour cells to treatment. We used two TP53-null neuroblastoma cell lines as in vitro models, and used siRNA to downregulate ITCH expression. Silencing of ITCH in vitro stabilizes TP73 protein on neuroblastoma cells and sensitizes the cells to irradiation treatment. Our results suggest that this novel strategy is feasible for combining with the conventional chemo-/ radio-therapy to treat the drug-resistant TP53-null neuroblastomas
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