Targeting RNA methyltransferase NSUN2 in epithelial ovarian cancer
Zorica Nakevska1, Farzaneh Afzali2, Zhihua Xu1, Tyler Cooper3, Rui Zhe Yang1, Holly Zhao1, Guihua Zhang1, DuPreez Smith4, James Key5, Gilles Lajoie3, Helen Steed4, Martin Köbel6, Cheng-Han Lee5, Lynne-Marie Postovit2, YangXin Fu1.
1Oncology, University of Alberta, Edmonton, AB, Canada; 2Biomedical & Molecular Sciences, Queen’s University, Kingston, ON, Canada; 3Biochemistry, Western University, London, ON, Canada; 4Obstetrics and Gynecology, University of Alberta, Edmonton, AB, Canada; 5Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada; 6Laboratory Medicine and Pathology, University of Calgary, Calgary, AB, Canada
Introduction. Dysregulated RNA modifications alter the epitranscriptome, resulting in aberrant mRNA expression and/or translation and tumorigenesis. RNA methyltransferase NSUN2 introduces 5-methylcytosine (m5C) to many types of RNAs and is implicated in cancers. Its role in epithelial ovarian cancer (EOC) remains elusive.
Methods. Association between NSUN2 expression and survival of EOC patients was analyzed using Kaplan-Meier (KM) plotter. The effect of NSUN2 knockdown (KD) and knockout (KO) on the behaviors and gene expression in EOC cells was determined by cell growth assay, western blotting, tumor xenograft model, as well as RNA-sequencing (RNA-seq) and proteomic analysis.
Results. High NSUN2 mRNA level is strongly associated with shorter overall survival of EOC patients. NSUN2 KD decreased cell growth in EOC cells, which is concurrent with increased expression of cell cycle inhibitor p27Kip1 and apoptosis-inducing protein PPIF and decreased expression of pro-survival protein Bcl-xL. However, the effect of NSUN2 KD became less pronounced over time, suggesting that adaptive mechanisms exist that help EOC cells compensate for the loss of NSUN2. In keeping with this, the effect of NSUN2 KO on cell growth was not as strong as NSUN2 KD in the early passages of cells. Preliminary experiments showed that NSUN2 KD and KO cells formed smaller tumors in mice, although it was not statistically significant. RNA-seq and proteomic analysis showed that NSUN2 KD and KO altered mRNA and protein levels of the genes associated with multiple biological processes, suggesting that the NSUN2-regulated epitranscriptome modulates gene expression in EOC cells.
Conclusions. Our results suggest a pro-tumorigenic role for NSUN2 in EOC. However, additional experiments are needed to characterize this role and the underlying molecular mechanisms. Furthermore, better understanding of the adaptive mechanisms that compensate for the loss of NSUN2 in EOC cells is warranted.
This research has been funded by the Alberta Women’s Health Foundation through the Women and Children’s Health Research Institute, and the Cancer Research Society and Ovarian Cancer Canada. .