Ubiquitin-specific proteases (USPs) are deubiquitinating enzymes, involved in the removal of ubiquitin from specific ubiquitinated substrates, often resulting in ‘escape’ from proteasomal degradation, and, regulation of protein localization or activation. USPs are potential therapeutic targets in cancer, due to their targetable cysteine protease function and their ability to regulate the stability of oncoproteins. USP5 is overexpressed in several malignancies, such as pancreas, ovarian, non-small lung cancer and myeloma, where it has been proposed as an oncogenic factor, promoting tumorigenesis by modulating cell cycle regulators. Most recently, the Project Achilles, which created a Cancer Dependency Map using the CRISPR/Cas-9 system, has identified neuroblastoma to have the highest dependency score for USP5, implicating USP5 may act as a key factor in neuroblastoma disease progression.
Neuroblastoma is the most common solid tumour in early childhood. Advanced neuroblastoma is associated with MYCN gene amplification and is currently fatal in 50% of children diagnosed with the disease. The aim of this project is to determine the oncogenic role of USP5 in MYCN-driven neuroblastoma. We have shown that knock-down of USP5 by specific siRNAs in MYCN-amplified neuroblastoma cell lines, led to a significant decrease in MYCN protein expression and half-life. Doxycycline induced repression of MYCN expression in SHEP-tet21N correlated with repressed USP5 protein and mRNA levels. Chromatin immunoprecipitation assays demonstrated a 2-7 fold enrichment of MYCN protein binding at canonical E Box regulatory sequences sited within the 500bp upstream USP5 gene promoter and within intron 1 of the USP5 gene in two neuroblastoma cell lines. USP5 was knocked-down in MYCN-amplified neuroblastoma cell lines, leading to a significant reduction of cell viability, cell proliferation, colony size and numbers in vitro. Taken together, our findings suggest USP5 and MYCN participate in a feedforward expression loop in neuroblastoma cells, ensuring high-level expression of both proteins, driving the disease. Inhibition of USP5 activity in neuroblastoma cells using small molecule inhibitors could be a potent novel therapy for MYCN-amplified tumours.