Abstract P4-02-06: Exploiting ceramide homeostasis to target endocrine therapy-resistant breast cancer
Authors: Pal Purab , Millner Alec , Atilla-Gokcumen Ekin , Frasor Jonna 
Published: March 2022

Abstract
Estrogen receptor (ER)-positive breast cancer accounts for the majority of breast cancer-related deaths. Current treatment includes endocrine therapy (ET), which most women respond to at first. However, up to 40% of women will eventually experience a relapse. Recurrent tumors tend to retain ER expression but no longer respond to ET. There have been multiple genomic and proteomic profiling studies between ET-sensitive and ET-resistant breast cancer cells, but lipidomic studies have been lacking. We have performed targeted and non-targeted lipidomic profiling of ET-sensitive and two models of ET-resistant MCF-7 breast cancer cells and found differences in their sphingolipid profiles, suggesting our overarching hypothesis that ceramide homeostasis is altered in ET-resistance. The current study is designed to understand the functional role of ceramides and their metabolites in ET-resistant breast cancer, with an overall goal to improve the current treatment regimens and quality of life in breast cancer patients. Detection of ceramides and other sphingolipid levels were analyzed by reverse-phase chromatography and LC-QToF-MS. C1P levels were detected by LC-QqQ-MS. Hoechst-PI staining was performed to measure cell death and Annexin-PI staining to detect apoptosis. All cytometric data analyses were performed using a Celigo cytometer. RT-qPCR was performed to analyze mRNA expression. Fold changes were calculated using the ΔΔCt method with multiple housekeeping controls. While numerous changes were observed in lipid species, the most pronounced changes were seen in the sphingolipidome. More specifically, ET-resistant breast cancer cells had lower levels of ceramides and di-hydroceramides. Along with a depletion of ceramides, we have also observed an increase in ceramide-1-phosphate (C1P) levels in the ET-resistant cells. C1P, which is produced by phosphorylation of ceramides by ceramide kinase (CERK), is known to promote cell survival and proliferation. This observation suggests that ET-resistant cells may rely on a higher degree of ceramide turnover to C1P compared to their ET-sensitive counterpart. To test this hypothesis, CERK was inhibited with a small molecule inhibitor (NVP-231), which resulted in increased apoptosis in ET-resistant cells but not in ET-sensitive cells, suggesting ET-resistant cells are more sensitive to CERK inhibition. Although NVP-231 treatment increased ceramide levels in both ET-sensitive and ET-resistant cells, only ET-resistant cells were dependent on ceramide turnover to C1P. To further understand the molecular underpinnings of the differential sensitivity to CERK inhibition, we have investigated the expression of several BCL2 family members, as evidence from the literature suggests that sphingolipids play a critical role in apoptosis by interacting with several BCL2 family members by mechanisms that are not thoroughly understood. We have observed a high positive correlation between BCL2 expression and NVP-231 sensitivity, as well as an inverse correlation between MCL-1 and NVP-231 sensitivity. This suggests that loss of BCL2 in the ET-resistant cells may be a factor for increased sensitivity to CERK inhibition and elevated MCL-1 fails to compensate for that loss. Together, our findings suggest that ET-resistant breast cancer cells have a unique ceramide homeostasis dependence, i.e., rely on a higher degree of ceramide turnover to C1P. Additionally, BCL2 family proteins may play a critical role in determining the sensitivity to CERK inhibition. The dependence of ET-resistant breast cancer cells on C1P production for survival can therefore offer a unique opportunity to increase the therapeutic efficacy and improve patient outcome for women with ET-resistant breast cancer.
Citation Format: Purab Pal, Alec Millner, Ekin Atilla-Gokcumen, Jonna Frasor. Exploiting ceramide homeostasis to target endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-06.