Biology subjects

Stebbing, J.

Publications and source records attributed to Stebbing, J..

5 recordsLinked to original sources

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↗

bioRxiv2023

CRISPR screens in 3D tumourspheres identified miR-4787-3p as a transcriptional start site miRNA essential for breast tumour-initiating cell growth

Breast cancer (BC) is a heterogeneous malignancy with distinct molecular subtypes and clinical characteristics. Tumour-initiating cells (TICs) are a small subset of cancer cells that are responsible for tumour initiation and progression. Our study employed pooled CRISPR screens, integrating 2D and 3D culture models, to identify miRNAs critical in BC tumorsphere formation. These screens combined with RNA-seq experiments allowed us to identify the miRNA signature and their targets that are essential for tumoursphere growth. Amongst them, miR-4787-3p exhibited significant up-regulation in BC, particularly in basal-like BCs, suggesting its association with aggressive disease phenotypes. Surprisingly, despite its location within the 5UTR of a protein coding gene, which define DROSHA-independent transcription start site (TSS)-miRNAs, our findings revealed its dependence on both DROSHA and DICER1 for maturation. Inhibition of miR-4787-3p hindered tumorsphere formation, highlighting its potential as a therapeutic target in BC. Moreover, our study proposes elevated miR-4787-3p expression as a potential prognostic biomarker for adverse outcomes in BC patients. We found that protein-coding genes positively selected in the CRISPR screens were enriched of miR-4787-3p putative targets. Amongst these identified key targets, we selected ARHGAP17, FOXO3A, and PDCD4 because are known tumour suppressors in cancer and experimentally validated the interaction of miR-4787-3p with their 3UTRs. Our work illuminates the molecular mechanisms underpinning miR-4787-3ps oncogenic role in BC. These findings advocate for further clinical investigations targeting miR-4787-3p and underscore its prognostic significance, offering promising avenues for tailored therapeutic interventions and prognostic assessments in BC.

cell biology↗

bioRxiv2020

Mutational landscapes of normal breast during age and pregnancy determine cancer risk

The accumulation of somatic mutations in the healthy breast throughout life and pregnancy is poorly understood1-10. Similarly, the mutational landscape of both epithelial and stromal components of the mammary gland has not been investigated. Both are relevant for breast cancer (BC), as the interplay between age, pregnancy, and cancer risk has not been fully characterized11. We describe whole genome sequencing analysis of epithelial and stromal compartments from the normal breast. We show that, in a similar way to other normal organs, the mutational burden of the mammary nulliparous epithelium significantly increases with age. In a nulliparous status, mutated clones are maintained at a consistently small size throughout the life of the individual; however, at parity, pre-existent clones significantly increase in size with age. Both epithelial and stromal compartments of the healthy breast contain pre-existing known cancer mutations, albeit at low rate, indicative of subsequent positive selection for mutations in tissue-specific driver genes. In line with this, both compartments also present gene enrichment in preferentially mutated cancer pathways. Our results show that mutational landscapes differ between the parous and nulliparous epithelium and suggest an explanation for both differential breast cancer risk and development of pregnancy-associated BC (PABC).

genetics↗

bioRxiv2019

Global kinome silencing combined with 3D invasion screening of the tumor microenvironment identifies fibroblast-expressed PIK3Cδ involvement in triple-negative breast cancer progression

As there is growing evidence for the tumor microenvironments (TME) role in tumorigenesis, we sought to investigate the role of fibroblast-expressed kinases in triple negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3C{delta} (f-PIK3C{delta}) as a key regulator of progression. Although PIK3C{delta} has been mainly described in leucocytes, we detected high expression in primary fibroblasts derived from TNBC patients, while PIK3C{delta} was undetectable in cancer epithelial cell lines. Genetic and pharmacologic gain- and loss-of functions experiments verified the contribution of f-PIK3C{delta} in TNBC cell invasion. By employing an integrated secretomics and transcriptomics analysis, we revealed a paracrine mechanism via which f-PIK3C{delta} confers its pro-tumorigenic effects. Inhibition of f-PIK3C{delta} promoted the secretion of factors, including PLGF and BDNF, which subsequently led to upregulation of NR4A1 in TNBC cells where it acts as a tumor suppressor. Inhibition of PIK3C{delta} in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3C{delta} in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, in addition to a decrease on tumor metastasis emphasizing the potential immune-independent effects of PIK3C{delta} inhibition. Finally, analysis of BC patient cohorts and TCGA datasets identified f-PIK3C{delta} (protein and mRNA levels) as an independent prognostic factor for overall and disease free survival, highlighting it as a therapeutic target for TNBC.

cancer biology↗