Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer
DNA methylation (5-methylcytosine, 5mC) is a major repressive epigenetic mark that can be iteratively oxidized by ten-eleven-translocation (TET) enzymes to 5-hydroxymethylcytosine (5hmC); beyond DNA, modifications of RNA can also affect transcript stability and processing, yet RNA hydroxymethylation remains unexplored in breast cancer (BC). Here, we identify a quantifiable cytoplasmic 5hmC signal in BC cell lines and, more prominently, in human BC tissue, using immunoassays and digital pathology, and confirm with an adapted, RNase-sensitive, RNA-specific ELISA that this signal reflects hydroxymethylation of total RNA and mRNA. Cytoplasmic 5hmC was significantly enriched in triple-negative (ER-/PR-/HER2-) and grade 3 tumors, and correlated with increased cytoplasmic TET2/TET3 in tissue and TET3 upregulation in TCGA. As a leading candidate mechanism, TET3 expression was associated with RNA-processing/spliceosomal transcripts, and TET3 knockdown in three TNBC cell lines reduced a subset of these targets, independently supported across 70 DepMap breast cancer lines.