Abstract P5-11-01: Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer
Authors: Semina Svetlana E. , Maienschein-Cline Mark , Alarid Elaine T. , Sartorius Carol A. , Frasor Jonna 
Published: March 2022

Abstract
Approximately 70% of breast cancers are estrogen receptor (ER) positive and can be treated with endocrine therapy. Unfortunately, 30-40% of tumors will recur, often as therapy-resistant metastatic disease. One contributor to disease recurrence is a population of cells with stem-like features (i.e. breast cancer stem cells, BCSCs). Previously, we have shown that positive crosstalk between two critical transcription factors, ER and NFκB, can contribute to the expansion of ER+ BCSCs, as indicated by increased mammosphere (MS) forming efficiency and BCSC markers. NFκB has been previously described as a major player in BCSC biology. However, the role of ER, as well as the interaction between ER and NFκB, in driving BCSCs remains unclear. We found in MS that both ER and NFκB are endogenously active and required for MS formation. To investigate whether ER and NFκB work together or independently, we made dual ERE and NFκB-RE reporter cell lines. We confirmed activation of both ER and NFκB in MS, but with a high degree of heterogeneity as indicated by 4 different cell populations (ERE+/NFκB-RE+, ERE+/NFκB-RE-, ERE-/NFκB-RE+ and ERE-/NFκB-RE-). Interestingly, we found &gt;90% of MS arose from ERE+ cells while NFκB-RE+ cells failed to from MS, suggesting that ER but not NFκB activity was sufficient to initiate MS development. Moreover, ERE+ cells, with or without NFκB-RE activity, formed more secondary MS and expressed higher levels of stem cell-associated genes than ERE- cells, indicating ERE+ cells are more stem-like. To study ER and NFκB function in more detail, we utilized single cell RNA sequencing of MS. We identified 4 clusters with clusters 1 and 2 enriched for stem cell gene signatures. Bioinformatic analysis revealed Cluster 2 to be enriched for ER, proliferation, and DREAM complex signatures, and we confirmed these genes were enriched in ERE+ cells. However, we also found that ER activity is associated with activation of the DREAM complex in standard cell culture. This suggests that ER regulation of proliferation via the DREAM complex may not be specific for BCSCs. In contrast to Cluster 2, Cluster 1 was enriched with ER and NFκB signatures, as well as an ER-NFκB crosstalk signature. In addition, Cluster 1 was enriched with pathways related to adaptive stress response such as: oxidative phosphorylation, unfolded protein response, and protein secretion. As some studies have reported activation of stress pathways is associated with maintenance of stemness, we hypothesize that ER and NFκB may work together to promote stem features through activation of multiple stress pathways to increase survival and growth in an anchorage-independent environment. To determine whether either Cluster 1 or Cluster 2 derived signatures are associated with clinical outcome, we first interrogated single cell RNA sequencing data from a metastatic ER+ PDX model. The Cluster 2 signature was enriched in proliferative cell populations found in both primary and metastatic tumors. In contrast, the Cluster 1 signature was enriched only in a metastatic tumor cell population. We also showed that expression of signatures derived from Cluster 1 or 2 in ER+ breast cancer patient tumors predict poor relapse free survival in the METABRIC dataset. However, only co-expression of both signatures predicted poor disease-free survival. Taken together, our findings suggest there are two unique subpopulations of stem-like cells, one of which is dependent on ER driven proliferation, whereas the other population relies on an ER-NFkB mediated stress response for survival and self-renewal. Furthermore, both of these population signatures in patients’ tumors are associated with more aggressive disease and poor outcome.
Citation Format: Svetlana E. Semina, Mark Maienschein-Cline, Elaine T. Alarid, Carol A. Sartorius, Jonna Frasor. Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ 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 P5-11-01.