Jonna Frasor

Up to 40% of patients with estrogen receptor positive breast cancer will experience relapse, either while on endocrine therapy (ET) or after ET is completed. A major contributor to ET failure is the presence of ET-insensitive cell populations within tumors. Here, we developed an analytical pipeline to systematically identify and target these populations by integrating single-cell RNA sequencing of ER+ tumors from the FELINE clinical trial with functional validation in a panel of patient-derived xenograft organoid models. We found that ET-insensitive cells are detected in all tumors regardless of clinical response and exhibit higher transcriptional heterogeneity than ET-sensitive populations. Using our pipeline, we identified and validated new therapeutic options that target patient-specific and shared ET-insensitive populations. Our integrated workflow provides a robust platform for identifying and targeting ET-insensitive cells and offers a translational framework to develop precision medicine approaches to improve outcome in breast cancer patients.

Figure S10. Kaplan-Meier plots of individual EnR-CIPs analyzed in METABRIC and kmPlotter databases.

Figure S1. Relative abundance of sphingolipids in multiple ET-resistant cell lines.

Despite the success of endocrine therapy (ET) in treating hormone receptor–positive breast cancer, a significant proportion of patients relapse during or after treatment, making ET resistance a major clinical challenge. Previously, we have shown that ET-resistant breast cancer cells exhibit reduced ceramide levels and an increased sensitivity to ceramide-induced cell death. In this study, we demonstrate that ceramides induce a distinct transcriptional reprogramming in ET-resistant cells, characterized by upregulation of endoplasmic reticulum stress (EnRS) pathways. Ceramide-induced EnRS is PERK-dependent and functionally linked to cell death in multiple models of ET resistance. Using a photoactivatable ceramide probe, we identify TRAM1 as a functionally important ceramide-interacting protein (CIP) in ET-resistant cells that correlates with worse relapse-free survival and a more aggressive breast cancer phenotype in patients with luminal breast cancer. Additionally, knockdown of TRAM1 phenocopies ceramide action in ET resistance, thereby suggesting its role in mediating ceramide-induced lethal actions in ET resistance. Together, our findings reveal that ET-resistant breast cancer cells are highly sensitive to PERK-mediated EnRS relative to ET-sensitive cells. Ceramides, likely via interactions with CIPs such as TRAM1, lead to PERK activation and consequential cell death in the ET-resistant breast cancer models. This sensitivity to ceramide-induced EnRS and cell death is a vulnerability that could be taken advantage of to treat ET-resistant breast cancer. Implications:This study elucidates the functional relevance of ceramide depletion in ET-resistant breast cancer cells.

Supplementary Table S10. Kaplan-Meier plots of individual EnR-CIPs analyzed in METABRIC and kmPlotter databases.

Figure S8. Validation of ceramide actions in PDxO models.

Figure S3. Validation of RNA-seq by RT-qPCR.

Supplementary Table S6 from Ceramide-Induced Endoplasmic Reticulum Stress Reveals a Targetable Vulnerability in Endocrine Therapy–Resistant Breast Cancer

Supplementary Table S5. Common upregulated and downregulated genes

Figure S2. Intracellular delivery of C8-ceramide.