Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer
- 1Department of Surgery and Cancer, Imperial College London, London, United Kingdom
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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BibTeX
@misc{wen2026cytoplasmic,
title = {{Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer}},
author = {J. Wen and L. Del Bel Belluz and S. Ottaviani and F. Poyia and H. Yu and S. Sachdev and L. El Eid and K. Garner and K. Mistry and E. Hayes and N. Moderau and P. C. Chandrasinghe and J. Stebbing and B. Cereser},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.21.760667},
url = {https://www.biorxiv.org/content/10.64898/2026.10.21.760667v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer
AU - J. Wen
AU - L. Del Bel Belluz
AU - S. Ottaviani
AU - F. Poyia
AU - H. Yu
AU - S. Sachdev
AU - L. El Eid
AU - K. Garner
AU - K. Mistry
AU - E. Hayes
AU - N. Moderau
AU - P. C. Chandrasinghe
AU - J. Stebbing
AU - B. Cereser
PY - 2026
DA - 2026/10/21
PB - bioRxiv
DO - 10.64898/2026.10.21.760667
UR - https://www.biorxiv.org/content/10.64898/2026.10.21.760667v1
AB - 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.
ER -