Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer

Wen, J.; Del Bel Belluz, L.; Ottaviani, S.; Poyia, F.; Yu, H.; Sachdev, S.; El Eid, L.; Garner, K.; Mistry, K.; Hayes, E.; Moderau, N.; Chandrasinghe, P. C.; Stebbing, J.; Cereser, B.

doi:10.64898/2026.10.21.760667

bioRxiv · 10.64898/2026.10.21.760667

Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer

Wen, J.·Del Bel Belluz, L.·Ottaviani, S.·Poyia, F.·Yu, H.·Sachdev, S.·El Eid, L.·Garner, K.·Mistry, K.·Hayes, E.·Moderau, N.·Chandrasinghe, P. C.·Stebbing, J.·Cereser, B.

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Abstract

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.

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Wen, J., Del Bel Belluz, L., Ottaviani, S., Poyia, F., Yu, H., Sachdev, S., El Eid, L., Garner, K., Mistry, K., Hayes, E., Moderau, N., Chandrasinghe, P. C., Stebbing, J., Cereser, B.. 2026-10-21. Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer. https://doi.org/10.64898/2026.10.21.760667

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