Abstract PS16-04: Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer
Authors: Semina Svetlana E. , Kastrati Irida , Cruz Luis Alejo , McCray Tara , Maienschein-Cline Mark , Alarid Elaine T. , Frasor Jonna 
Published: February 2021

Abstract
Approximately 50% of estrogen receptor (ER) positive breast cancer patients will experience relapse. One explanation for this is the presence of breast cancer stem cells (BCSCs), which have self-renewal capability and can contribute to therapy resistance, metastasis, and relapse. Previously, we have shown that cells with active ER and NFκB gain stem cell properties. They are characterized by higher mammosphere (MS) forming efficiency and increased expression of stem-associated markers. NFκB has been previously described as a player in BCSC biology. However, the role of ER remains unclear since some studies have suggested that BCSCs are ER negative. In order to study the role of ER and NFκB in BCSCs in more detail, we utilized single cell RNA sequencing of MS. Dimensionality reduction of single cells identified 4 clusters with unique transcriptional signatures. In order to determine which cluster of these 4 represents BCSCs, we ran functional enrichment analysis (FEA) using MsigDB as a source of various stem cell signatures. Results of FEA showed 1 of 9 stem cell signatures enriched in Cluster 0. Whereas clusters 1, 2 and 3 demonstrated increasing enrichment of different stem cell signatures, respectively. Further analysis of stem cell signatures and genes revealed that cluster 1 included not only classical stem markers (ALDH1A3, KRT8, CD36), but also was enriched for PI3K and p53 signaling pathways. The cluster 2 stem cell signature was associated with DREAM complex activity and BRCA1 mutation. Cluster 3 was enriched for expression of NANOG and SOX2 targets. We also ran FEA to determine ER and NFκB activity in each population. Cluster 0 showed no activity of either ER or NFκB, whereas Cluster 1 was enriched for both, cluster 2 was enriched for ER only, and cluster 3 was enriched for NFκB only. Taken together this data suggests that ER and NFκB may play different roles in promoting BCSC properties. In order to determine how both pathways effect BCSC features, we made a dual reporter cell line expressing ERE-mCherry and NFκB-RE-GFP. It was found ERE activity was constant throughout MS formation, whereas NFκB-RE activity increased proportionally to MS growth over time, suggesting these pathways have different dynamics and further supporting unique roles for each in MS development. To determine which cell population has the capacity to seed MS, an indication of stemness, we sorted cells by reporter activity and tested MS forming efficiency. We discovered that ERE+ cells, either with or without NFκB activity, are more stem-like, whereas activation of NFκB alone is not sufficient to drive MS development. This finding was verified by stem cell-associated gene expression. These data suggest that ER activity is a key driver for BCSC properties and that NFκB may play a supporting role. Furthermore, a novel mechanism of ER regulating the DREAM complex, which is known to be involved in cancer progression and used to evaluate the risk of recurrence in breast cancer, is suggested as a potential ER-mediated mechanism for driving BCSCs. Taken together, our findings suggest novel roles for ER and NFκB in BCSC biology, which could be exploited to target BCSCs therapeutically in ER+ breast cancer patients in order to improve their outcome.
Citation Format: Svetlana E. Semina, Irida Kastrati, Luis Alejo Cruz, Tara McCray, Mark Maienschein-Cline, Elaine T. Alarid, Jonna Frasor. Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-04.