Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells

SIMPLE SUMMARY: Endocrine therapy (ET) resistance is a major problem in estrogen receptor-positive breast cancer patients. Since there have been few lipidomic studies in ET resistance and sphingolipids are heavily implicated in multidrug-resistant and chemotherapy-resistant cancers, we aimed to investigate the sphingolipidome of tamoxifen-resistant breast cancer cells in search of a unique sphingolipid profile that can potentially be exploited therapeutically. We found that ET-resistant breast cancer cells maintain a lower level of ceramides for their survival. In order to achieve this, they are dependent on ceramide kinase (CERK), the activity of which helps maintain low endogenous ceramide levels, therefore promoting tamoxifen-resistant cell survival. Targeting CERK can therefore represent an opportunity to target therapy-resistant breast tumors and improve the patient outcome for women with ET-resistant disease. ABSTRACT: ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.