Ubiquitin Carboxyl‐Terminal Esterase L1 (UCHL1) Regulates Stem‐Like Cancer Cell Populations in Pediatric High‐Grade Glioma
Authors: Sanchez Diaz Patricia C , Chang Judy C , Dao Tu , Chen Yidong , Hung Jaclyn Y 
Published: November 2023

Background and Significance

Pediatric high‐grade gliomas (HGG) represent 8–12% of all primary tumors of the nervous system in children. Five‐year survival for these pediatric aggressive tumors is poor (15–35%) indicating the need to develop better treatments for pediatric HGG. Cancer stem cells (CSCs) represent a highly tumorigenic subpopulation within the tumor responsible for its relapse. Depicting the pathways that regulate maintenance of these CSCs may lead to novel therapies and improved patient outcomes. In this work we used cell lines to study the function of the ubiquitin carboxyl‐terminal esterase L1 (UCHL1), a deubiquitinase de‐regulated in some cancers, in pediatric high‐grade glioma CSCs.

Methods

SF188 cell line was kindly provided by Dr. Daphne Haas‐Kogan of the University of California at San Francisco. SJ‐GBM2 was obtained from the Children's Oncology Group (COG) Cell Culture and Xenograft Repository. Stable UCHL1 knockdowns (KDs) were obtained through a lentiviral transduction system (Addgene) using puromycin as marker for selection. Two different shRNA constructs targeting UCHL1 coding region were used. Cells infected with shTurboGFP vector were included as control in all experiments. Cell invasiveness was measured using CytoSelect 24‐Well Cell Invasion Assay (Cell Biolabs). Sphere formation was used as CSCs read‐out assay. One‐way ANOVA (SPSS, IBM) was used to determine statistical significant differences in these in vitro assays. RNAseq (Illumina) and Ingenuity Pathway Analysis (Qiagen) were conducted to identify UCHL1‐associated gene networks.

Results

UCHL1 depletion caused an impairment in sphere formation (65% and 90% reduction in SF188 and SJ‐GBM2 respectively) compared to the cells expressing the control shRNA construct (p<0.01). A 90% decrease in invasiveness was also observed in all the UCHL1 KDs (p<0.001). Preliminary transcriptome comparisons of UCHL1 KD versus vector identified approximately 300 differentially expressed genes including: ACTA2, FBXL7, FBXW11, LIF, POSTN, GDF15, HMOX1, KIT, RAC1, WNT5A, PIK3R1, SOX5, PIP5K1C, NURP1, CDKN1A, AQP4, AQP3, CXCL12, DLL1, DLK1, DLX4, ITGA11, ITGB8, MTSS1, and TLR4. In silico gene ontology analysis of the identified genes revealed significant enrichment of the processes involved in cell adhesion, invasion, and motility. In addition, and using a TCF‐dependent TOPflash reporter activity assay, we found a 30% reduction in Wnt signaling activity in the UCHL1 knockdowns compared to controls.

Conclusions

UCHL1 inhibition impaired invasiveness and sphere formation in pediatric high‐grade glioma cell lines. Gene expression and bioinformatic analyses identified cell adhesion, invasion, and motility as potential biological processes regulated by UCHL1. Our results suggest that UCHL1 may regulate CSC self‐renewal in pediatric HGG and that targeting UCHL1 might complement current therapeutic regimens.

Support or Funding Information

Funding for this work was provided in part by the Greehey Cancer Research Institute Permanent Health Fund Permanent Endowments – Higher Education (Children's Cancer Research) at the University of Texas Health Science Center at San Antonio, the Greehey Graduate Fellowship in Children's Health (Greehey Family), the Shelby Rae Tengg Foundation, and the University of the Incarnate Word Faculty Development Funds. TD was a student in the 2015 RSO Summer Fellowship Training Program.