Abstract PS3-02-08: Medium-chain fatty acid exposure in non-transformed mammary glands leads to pro-tumorigenic alterations associated with aging

Abstract Introduction. The local breast environment is an important source for identifying the etiological and biological factors that contribute to the development of breast cancer (BC). We identified a lipid metabolism gene signature associated with Estrogen Receptor negative BC (ERnegBC) in high-risk benign breast tissue. We then exposed non-transformed breast cells and breast microstructures in vitro and ex vivo to fatty acids (FA) and observed a metabolic shift toward the serine, one-carbon, glycine and methionine pathways, engendering epigenetic plasticity, increasing reactive oxygen species (ROS), and promoting cell survival. This promted the question: are there in vivo context(s) where lipid alterations resemble those of FA exposure in vitro/ex vivo? Since epigenetic plasticity and increased ROS are also reported in the aged mammary gland, we hypothesize that FA-induced metabolic reprogramming increases in the aged mammary gland, contributing to pro-tumorigenic alterations observed during aging. Methods. MCF-10A cells were exposed to the medium-chain FA octanoic acid (OA) for proteomics. Breast tissue-derived microstructures exposed to ± OA were utilized for single-cell RNA-seq. Breast microstructures and 3D mammary spheres (formed from mammary cells in Matrigel) were exposed to ± OA for 7 days, stained for luminal/basal markers, F-actin, and nuclei, and imaged using confocal microscopy. Raman spectroscopy was used to characterize the lipid content in pre- versus post-menopausal breast tissue. Results. In vitro and ex vivo data on FA exposure in non-transformed mammary cells and breast microstructures revealed striking similarities to the aged mammary gland: 1) Significant (p < 0.01) aging-related changes in gene expression upon OA exposure in epithelial and non-epithelial compartments. Notably, up-regulation of GDF15 (related to EMT, invasion, lipolysis and aging), MDK (involved in neurogenesis, cancer progression, and aging) and the downregulation of MMP7, a gene for which reduced expression contributes to the accelerated aging of mammary epithelial cells. Moreover, OA led to significant downregulation (p < 0.01) of lineage markers. 2) Significant (p < 0.01) upregulation of components of the Senescence-Associated Secretory Phenotype in both epithelial and non-epithelial subtypes. Including those that promote reprogramming (AREG), cancer progression (EREG), EMT (CCL20) and migration (ANGPTL4). 3) Altered inter-cellular communications upon OA exposure. Cell-cell communication analysis indicated increased secreted signaling in OA, with AREG, GDF15, and MDK among the strongest signals. 4) Decreased cell-cell junctions and extracellular matrix-receptor (ECM-R) interactions. Differential proteomics revealed downregulation of proteins involved in ECM-R interaction and focal adhesion. Cell-cell communication analysis also revealed decreased ECM-R interactions following OA exposure, alongside reduced cell-cell adhesions. 5) OA led to aging-associated pro-tumorigenic changes that may increase susceptibility to BC: Ex vivo culture of breast microstructures and 3D mammary spheres revealed that OA exposure alters cell-cell adhesions, compromising the basal barrier and leading to luminal cell dissemination. Conclusions. The proportion of breast adipocytes releasing free FAs increases with age. We show that FA exposure causes changes typical of the aged mammary gland, such as elevated ROS, epigenetic alterations, downregulation of cell-cell junctions, altered ECM-receptor interactions, and aging associated gene changes. We propose that the release of free FAs from an increased number of breast adipocytes (potentially due to elevated GDF15-induced lipolysis during aging) is a factor that induces mammary gland remodeling, increasing vulnerability to BC. Citation Format: M. Bustamante Eduardo, A. B. Islam, M. Zappia, A. Z. Samuel, C. W. McCloskey, M. V. Frolov, R. W. Khokha, E. V. Benevolenskaya, S. A. Khan, S. E. Clare. Medium-chain fatty acid exposure in non-transformed mammary glands leads to pro-tumorigenic alterations associated with aging [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-02-08.