Neuroblastoma (NB) is a pediatric malignancy derived from the sympaticoadrenal lineage of neural crest. It is the most commonly occurring extra-cranial nervous system malignancy of infants and children under the age of 5. Long-term survival is less than 40% in patients with high risk NB and over half of the survivors relapse. Emerging evidence supports the notion that there exists within tumors a small population of cells exhibiting stem-like properties, termed cancer stem-like cells (CSC), that is responsible for clinical relapse; therefore, investigating CSC targets may lead to improved treatments. Recent studies have demonstrated that deubiquitinating enzymes (DUBs) of the ubiquitin proteasome system are important players in cancer-associated pathways. These enzymes could be potential effective therapeutic targets. Previous work has identified expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1), a DUB, in the CSC pool of neuroblastoma cells. Our studies are focused on the potential pathways and mechanism by which UCHL1 is acting in the CSC pool. We performed in vitro analyses on NB cell lines SK-N-BE(2), SK-N-AS, and SK-N-DZ. These cells differ in their MYCN amplification and P53 status. The CSC pool was enriched using the sphere formation assay. The small molecule inhibitor, LDN-57444, was used to impair UCHL1 activity. Furthermore, site-directed mutagenesis was used to generate the C90S mutant, which also blocks UCHL1 activity. The effect of blocking UCHL1 activity on cellular proliferation and CSC self-renewal was determined by the MTS assay and sphere formation assay respectively. We further confirmed the effect of impairing UCHL1 activity on the CSC population using flow cytometry by assessing the CD133 and side population. The soft agar assay was used to study the anchorage independent growth of UCHL1 impaired cells. Droplet digital PCR was used to investigate the effect of impaired UCHL1 activity on genes in the Wnt signaling pathway. Immunoprecipitation assays were used to confirm presence of C90S mutant in transfected cells and to determine potential interaction between UCHL1 and Wnt signaling proteins such as β-catenin. UCHL1 inhibition with LDN-57444 demonstrated a dose-dependent decrease in cellular proliferation and impaired sphere formation capability (read-out for self-renewal). We observed differential gene expression levels of MYC and MYCN in cells with UCHL1 inhibited activity via LDN-57444 treatment. C90S mutant transfected cells also demonstrated impaired self-renewal. C90S mutant cells showed decreased gene expression of Wnt signaling proteins and differential expression of MYC and MYCN. Immunoprecipitation studies and CD133 and side population analyses for assessing the CSC population are ongoing. Our data indicates that UCHL1 is potentially an important player in the regulation of CSC self-renewal. Our results also suggest that UCHL1 may mediate self-renewal through the MYC family of transcription factors. The MYC family has been shown to be promising targets in NB. Reduced MYC or MYCN gene expression in NB leads to cell cycle arrest and/or apoptosis. Further investigation of the effect of UCHL1 impaired activity on Wnt signaling will clarify whether UCHL1 is affecting MYC transcription through this pathway. Our studies indicated that UCHL1 might potentially be an effective therapeutic target for the CSC population to supplement current treatments.
Citation Format: Judy C. Chang, Jaclyn Y. Hung. Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B49.