RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m 5 C), in the transcriptome remains unclear. Aberrant regulation of 5-methylcytosine turnover in cancer may affect posttranscriptional modifications in coding and noncoding RNAs in disease pathogenesis. Mutations in NSUN2 have been reported as drivers of neurodevelopmental disorders in mice, and upregulated expression of NSUN2 in tumors of the breast, bladder, and pancreas has been reported. In this study, we conducted mRNA whole transcriptomic bisulfite sequencing to categorize NSUN2 target sites in the mRNA of human pancreatic cancer cells. We identified a total of 2829 frequent m 5 C sites in mRNA from pancreatic cancer cells. A total of 90.9% (2572/2829) of these m 5 C sites were mapped to annotated genes in autosomes and sex chromosomes X and Y. Immunohistochemistry staining confirmed that the NSUN2 expression was significantly upregulated in cancer lesions in the LSL-Kras G12D/+ ;Trp53 fl/fl ;Pdx1-Cre (KPC) spontaneous pancreatic cancer mouse model induced by Pdx1-driven Cre/lox system expressing mutant Kras G12D and p53 deletion. The in vitro phenotypic analysis of NSUN2 knockdown showed mild effects on pancreatic cancer cell 2D/3D growth, morphology and gemcitabine sensitivity in the early phase of tumorigenesis, but cumulative changes after multiple cell doubling passages over time were required for these mutations to accumulate. Syngeneic transplantation of NSUN2-knockdown KPC cells via subcutaneous injection showed decreased stromal fibrosis and restored differentiation of ductal epithelium in vivo. Significance Transcriptome-wide mRNA bisulfite sequencing identified candidate m 5 C sites of mRNAs in human pancreatic cancer cells. NSUN2-mediated m 5 C mRNA metabolism was observed in a mouse model of pancreatic cancer. NSUN2 regulates cancer progression and epithelial differentiation via mRNA methylation.