Abstract 5296: Epigenetic and transcriptional drivers of mucinous breast carcinoma

Abstract Background: Mucinous breast cancer (MucBC) is an uncommon histologic subtype of estrogen receptor (ER)-positive/HER2-negative breast cancer (BC) characterized by tumor cells floating in pools of mucin. In contrast to ER-positive/HER2-negative invasive ductal carcinoma of no special type (IDC-NST), MucBCs harbor fewer PIK3CA mutations and typically lack concurrent 1q gains/16q losses. Yet, no pathognomonic genetic alterations have been identified to explain their mucinous phenotype. Here, we sought to determine whether MucBCs might be driven by distinctive epigenetic alterations. Methods: We analyzed 40 pure MucBCs using genome-wide DNA methylation profiling (n=40) and/or RNA-sequencing (n=27). We evaluated DNA methylation age, epigenetic mitotic score, and integrated methylation-transcriptomic profiles, comparing MucBCs with IDC-NSTs from The Cancer Genome Atlas (TCGA) lacking PIK3CA mutations and concurrent 1q gains/16q losses to minimize genetic confounding factors. IDC-NSTs were matched at a 1:1 ratio by menopausal status, ER/HER2 status and histologic grade. The ER-positive/HER2-negative CAMA1, ZR-75-1 and MCF7 breast cancer cell lines were used to interrogate candidate pathways and transcriptomic changes. Results: Compared to matched IDC-NSTs, MucBCs exhibited accelerated DNA methylation age relative to patient chronological age (p=0.015), indicative of epigenetic dysregulation, lower epigenetic mitotic scores (p=0.0071), and pronounced global enhancer hypomethylation (p 0.001). Integrated methylation-transcriptome analyses identified ZBTB20 and ZNF133 as key transcriptional regulators in MucBCs (FDR 0.05), as well as significant dysregulation of the TGF-β and estrogen-response pathways (FDR0.05). In CAMA1 and ZR-75-1 BC cells, pharmacologic inhibition of the TGF-β pathway led to increased expression of mucin-encoding genes (MUC2, MUC20, MUCL1, MUC5B), while silencing of ZBTB20 or ZNF133 resulted in reduced mucin gene expression. In MCF7 cells, overexpression of ZBTB20 or ZNF133 similarly resulted in upregulation of mucin encoding genes and induced transcriptional changes in the TGF-β pathway, including downregulation of TGFBR1, TGFB1, SMAD3 and TRIM33 (TIF1γ) and upregulation of ID1, mirroring patterns observed in MucBCs. Conclusions: MucBC display accelerated DNA methylation age and widespread enhancer hypomethylation, underscoring profound epigenomic dysregulation. Key transcription factors, including ZBTB20 and ZNF133, appear to orchestrate mucin gene expression as well as TGF-β pathway reprogramming, contributing to the mucinous phenotype of MucBC. Citation Format: Lorenzo Ferrando, Higinio Dopeso, Laxmi Gusain, Edaise M da Silva, Thais Basili de Oliveira, Lounes Djerroudi, Hannah Y. Wen, Hong Zhang, Edi Brogi, Richard P. Koche, Pierre-Jacques Hamard, Larry Norton, Jorge S. Reis-Filho, Britta Weigelt, Fresia Pareja. Epigenetic and transcriptional drivers of mucinous breast carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5296.