Ottaviani, S. | Search bioRxiv

bioRxiv2026

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.

cancer biology↗

bioRxiv2025

Single-cell transcriptomics uncovers that cell-to-cell communication controls p53 activation

Conventional chemotherapy remains a cornerstone of cancer treatment and is highly effective in the adjuvant and neoadjuvant settings. However, therapeutic resistance, especially in the metastatic context, limits its long-term efficacy. While significant progress has been made in elucidating mechanisms of chemotherapy resistance, the role of cell-to-cell communication in modulating treatment response remains largely unexplored. Here, we combined microfluidic technology with single-cell RNA sequencing (scRNA-seq) to directly compare the transcriptional responses of cells in communication versus those in isolation. Upon dox treatment, we observed robust activation of the p53 pathway in communicating cells, whereas this response was markedly impaired in isolated cells. Moreover, pharmacological inhibition of caveolar-mediated endocytosis mimics the loss of p53 activation, further implicating this pathway in the communication-dependent regulation of chemotherapy response. This finding uncovers a previously unrecognized and essential role for paracrine signalling in mediating p53-dependent responses to chemotherapy. Our study highlights the critical importance of cell-to-cell communication in shaping therapeutic outcomes and opens new avenues for understanding and potentially overcoming chemotherapy resistance.

cancer biology↗