Abstract P4-12-22: Ergosterol Peroxide affects the VCP/ANKZF1 complex spatial relationship and interaction in IBC cells

Abstract Inflammatory breast cancer (IBC) is a highly aggressive and lethal type of breast cancer. Furthermore, patients with the triple-negative breast cancer subtype have tumors that lack the estrogen receptor, progesterone receptor, and HER2 expression. These tumors display greater invasiveness, and consequently, patients exhibit increased metastasis. Due to the lack of targeted therapies, IBC patients typically undergo multimodality therapy with cytotoxic chemotherapy like taxanes and anthracyclines, radiation, and surgery. There is an urgent need for selective therapies for IBC and natural products have gained significant attention for their potential in breast cancer therapy. In our laboratory, we work with Ergosterol Peroxide (EP), a bioactive compound extracted from the Ganoderma lucidum mushroom. Our studies indicate that EP is a unique endoperoxide that upon entrance into the cancerous cell, deploys its warhead via selective interactions with intracellular proteins, without any effect in non-cancerous cells. The Valosin-containing protein (VCP) / Ankyrin repeat and zinc finger domain-containing protein 1 (ANKZF1) complex plays a vital role in maintaining cellular proteostasis and ensuring proper protein synthesis and degradation processes. ANKZF1 interacts with VCP and translocates to the mitochondria under cellular stress conditions. We believe that EP affects this complex to induce cancer cell death. We hypothesize that EP targets the VCP/ANKZF1 complex, impairing the ability of IBC cells to clear damaged proteins and causing mitochondria dysfunction to induce IBC cell death. To validate the interruption of VCP/ANKZF1 interactions, we used Proximity Ligation Assays (PLA) with antibodies for VCP and ANKZF1 in the IBC cell model, SUM149. Veh, EP, and NMS 873 (VCP inhibitor, positive control) were administered, to detect changes in PLA signals for VCP and ANKZF1. To understand the VCP/ANKZF1 spatial relationship, under Veh, EP, and H2O2 (positive control) effects in SUM149, we used a colocalization analysis by Immunocytochemistry (ICC). Results were visualized with Gen 5 Image Prime 3.15 program in Cytation C10 Confocal Imaging Reader. The PLA preliminary results indicate that EP affects protein-protein interactions, since the intensity of green fluorescence, which indicates protein activity, is lower in the treated cells. Especially when compared to the positive control - NMS 873 the protein inhibitor. The preliminary results of ICC demonstrated that when SUM149 cells are treated with EP compound, VCP displays greater fluorescence intensity, while ANKZF1 fluorescence is diminished. In addition, cells treated with EP display a nucleus with greater size when compared to the Veh. Furthermore, ANKZF1 looks to be co-localizing with the nucleus, while VCP has an around and near localization of the nuclei when compared with the Veh. In general, EP affects VCP/ANKZF1 spatial relationship and interaction in IBC cells. Further research is needed to comprehensively understand the intricate mechanisms through which EP exerts its antineoplastic effects in IBC cells. Citation Format: Luz V. Arroyo-Cruz, Adriana Y. Aponte-Ramos, Michelle M. Martínez-Montemayor. Ergosterol Peroxide affects the VCP/ANKZF1 complex spatial relationship and interaction in IBC cells [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-22.