Abstract 4757: Elucidating novel pro-apoptotic consequences of ceramides in endocrine therapy-resistant breast cancer

Abstract Endocrine Therapy (ET)-resistant breast cancer (BC) remains a clinical problem, with approximately 40% of patients experiencing disease relapse. Previously, we demonstrated that preclinical models of ET-resistant BC have an altered sphingolipidome, which is characterized by low levels of ceramides across multiple models of ET resistance. ET-resistant models are also more sensitive to ceramide-induced death compared to their ET-sensitive counterparts suggesting a functional significance of the low levels of ceramides. This study is designed to identify mechanisms by which ceramides preferentially kill ET-resistant BC models to identify novel therapeutic strategies to improve ET-resistant BC patient outcomes. We performed total RNA-seq of multiple ET-resistant BC cells with or without ceramides and found that ceramide exclusively induces genes associated with endoplasmic reticulum stress (EnRS), particularly in the PERK/peIF2A/ATF4 pathway, in multiple models of resistance but not in the parental ET-sensitive cells. Inhibitors of EnRS, PERK, and peIF2A partially reverse ceramide-induced cell death suggesting that ceramide-induced EnRS is lethal for ET-resistant cells. Since ceramides are well-known bioactive lipids, we hypothesized that ceramides interact with specific proteins that are instrumental in ET-resistant cell survival. To test this, we used a photoactivable-and-clickable ceramide probe and quantitative proteomics to identify differentially expressed ceramide-interacting proteins (CIPs) in ET-resistant BC cells. We found that ceramide interacts with a group of clinically relevant CIPs which are located in the endoplasmic reticulum (EnR) membrane. While most of these CIPs have yet to be implicated in ET resistance, a subset of the CIPs is known to be involved in attenuating EnRS. Knocking down one of the top CIPs, i.e., TRAM1, phenocopies ceramide treatment, suggesting that ceramides, by interacting with EnR-membrane proteins, such as TRAM1, may disrupt their function and induce a lethal level of EnRS in ET-resistant cells. Together, we find that ceramide induces preferential cell death in ET-resistant cells through interactions with a novel array of proteins leading to the inhibition of key survival proteins and a consequential activation of lethal levels of EnRS. Understanding how ceramides interact with and affect these proteins could lead to the development of novel strategies to selectively treat ET-resistant BC and to improve patient outcomes. Citation Format: Purab Pal, Godwin K. Sarpey, G. Ekin Atilla-Gokcumen, Jonna Frasor, Jonathan L. Coloff. Elucidating novel pro-apoptotic consequences of ceramides in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4757.