title: Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.
abstract: Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.
authors.affiliation: Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Sartorius
authors.affiliation: Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: C T
authors.initials: CT
authors.lastname: Hanna
authors.affiliation: Women's Malignancies Branch, National Cancer Institute, Bethesda, MD, USA.
authors.forename: B
authors.initials: B
authors.lastname: Gril
authors.affiliation: Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: H
authors.initials: H
authors.lastname: Cruz
authors.affiliation: Department of Anesthesiology, University of Colorado Denver, Anschutz Medical, Aurora, CO, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Serkova
authors.affiliation: Department of Anesthesiology, University of Colorado Denver, Anschutz Medical, Aurora, CO, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Huber
authors.affiliation: Department of Medicine, Division of Medical Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: P
authors.initials: P
authors.lastname: Kabos
authors.affiliation: Department of Medicine, Division of Medical Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: T B
authors.initials: TB
authors.lastname: Schedin
authors.affiliation: Department of Medicine, Division of Medical Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: V F
authors.initials: VF
authors.lastname: Borges
authors.affiliation: Women's Malignancies Branch, National Cancer Institute, Bethesda, MD, USA.
authors.forename: P S
authors.initials: PS
authors.lastname: Steeg
authors.affiliation: Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Cittelly
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000473:pathology*; D001932:Brain Neoplasms / Q000556:secondary*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002471:Cell Transformation, Neoplastic; D066246:ErbB Receptors / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D019899:Paracrine Communication / Q000187:drug effects; D064726:Triple Negative Breast Neoplasms / Q000473:pathology*
chemical_list: D004967:Estrogens; D004958:Estradiol; D066246:ErbB Receptors