Abstract P4-04-04: Estrogen signaling through astrocytes promotes migration and invasion of ER-negative brain metastatic breast cancer cells
Authors: Cittelly Diana M , Cruz Hazel , Serkova Natalie J , Virginia Borges F , Peter Kabos , Patricia Steeg S , Carol Sartorius A 
Published: May 2015

Abstract
Approximately 16% of patients with breast cancer develop symptomatic brain metastases (BMs) and the majority [∼80%] will die subsequently. Symptomatic BMs are more prevalent in breast tumors overexpressing Human Epidermal Growth Factor Receptors (HER1, EGFR) or 2 (HER2) and triple-negative (TN) tumors (lacking estrogen receptor (ER), progesterone receptor (PR) and HER2). Although the majority of breast cancer cells colonizing the brain lack ER, central nervous system (CNS) metastases are more frequent in pre-menopausal women with high levels of circulating estrogens. Since astrocytes are ER+ cells that surround and infiltrate brain metastases, we hypothesize that estrogens act in a paracrine manner on reactive astrocytes to promote BMs.
Results: To determine the effect of estrogen in brain colonization, a brain-seeking sub-line of human MDA-MB-231 TN breast carcinoma cells (231Br) cells were injected intracardially in ovariectomized female nude mice supplemented with placebo (n=5) or 1mg 17-βestradiol (E2) pellets (n=5), and metastases were detected by gadolinium-enhanced magnetic resonance imaging (MRI) 5 weeks later. 100% (5/5) of mice from E2 group showed large and multiple BM when compared to 3/5 (60%) of placebo mice. Further, E2-treated mice showed decreased survival (41.6%, n=12) by 3 weeks following IC injection of 231Br cells compared to E2-depleted mice (84%, n=12) (P=0.03). Since astrocytes are the most abundant brain cell type expressing ERs, we focused on their interaction with 231BR cells. Using global gene expression arrays and RT-PCR we found that E2 upregulated Egf and Tgf-a in primary mouse astrocytes and human astrocytes (derived from neural stem cells), and that anti-estrogens blocked this effect. Concentrated conditioned media (CM) from E2-treated astrocytes increased P-EGFR levels in 231Br cells compared to vehicle (EtOH), or E2+ICI astrocytic CM. Moreover, CM from E2-treated astrocytes significantly increased migration and invasion of 231Br cells as compared to EtOH-treated CM, and ERs inhibitors abolish this effect. Treatment of 231Br cells with lapatinib abolished EGFR activation in response to E2-astrocytic CM, and decreased migration and invasion of 231BR cells. These data suggest that EGFR-ligands upregulated by E2 in astrocytes activated brain metastatic cells-EGFR resulting in increased migration and invasion. To identify the mechanisms of increased migration and invasion we performed global gene expression profiling of 231Br cells co-cultured with E2- or EtOH-stimulated-astrocytes. Co-culture with E2-treated astrocytes significantly increased expression of metastatic mediators Matrix-metalloproteinase-9 (MMP9) and S100 Calcium-binding protein A4 (S100A4) in 231Br cells, and treatment of astrocytes with ER inhibitors abolished this effect. Furthermore, MMP9 or S100A4 knockdown in 231Br cells significantly decreased migration and/or invasion in response to E2-astrocytic CM, suggesting that MMP9 and S100A4 are functional mediators of the paracrine effect of E2. Conclusion: These studies suggest that EGFR activation by astrocytic ligands is at least one mechanism by which E2 contributes to the promotion of BM of ER- breast cancer cells and suggests a novel role for ER in breast cancer BM.
Citation Format: Diana M Cittelly, Hazel Cruz, Natalie J Serkova, Borges F Virginia, Kabos Peter, Steeg S Patricia, Sartorius A Carol. Estrogen signaling through astrocytes promotes migration and invasion of ER-negative brain metastatic breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-04.