title: Dynamic monitoring of GPER-mediated estrogenic effects in breast cancer associated fibroblasts: An alternative role of estrogen in mammary carcinoma development.
abstract: Cancer associated fibroblasts (CAFs) are crucial contributors to breast cancer development. Estrogen affects mammary stroma in both physiological and pathophysiological conditions. We show here that estrogen (G-protein coupled) receptor (GPER) could be detected by immunohistochemistry in stromal fibroblasts of primary breast cancers. The presence of GPER expression was further confirmed by immunofluorescence and quantitative PCR in CAFs isolated from primary breast cancers. Based on dynamic monitoring by real time cell analyzer (RTCA) system, 17-β-estradiol (E2) as well as GPER specific agonist G1 were observed to trigger transient cell index increasing within an hour in a dosage-dependent manner in breast CAFs. In addition, E2 and G1 stimulated intracellular calcium modulation and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 within seconds and minutes in CAFs, respectively. Moreover, E2 and G1 promoted cell proliferation of breast CAFs measured by RTCA monitoring, cell viability assay and cell cycle analysis, and this promotion could be blocked by a GPER-selective antagonist G15. Interestingly, dynamic RTCA monitoring indicated that E2 increased adhesion of resuspended cells, and microscopy confirmed that E2 stimulated cell spreading. Both the adhesion and spreading were proposed to be mediated by GPER, since G1 also stimulated these effects similar to E2, and G15 reduced them. Moreover, GPER was found to mediate migration that was increased by E2 and G1 but reduced by G15 in RTCA cell migration assay and transwell assay. Accordingly, GPER mediates not only rapid actions but also slow effects including adhesion/spreading, proliferation and migration in breast CAFs. Estrogen is likely to affect tumor associated stroma and contributes to mammary carcinoma development through CAFs.
authors.affiliation: Department of Thyroid and Breast Surgery, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
authors.forename: Haojun
authors.initials: H
authors.lastname: Luo
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Manran
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Gynecology and Obstetrics, Chongqing Health Center for Women and Children, Chongqing 400010, China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Luo
authors.affiliation: Department of Endocrine and Breast Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Tenghua
authors.initials: T
authors.lastname: Yu
authors.affiliation: Department of Endocrine and Breast Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Chengyi
authors.initials: C
authors.lastname: Wu
authors.affiliation: Department of Endocrine and Breast Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Guanglun
authors.initials: G
authors.lastname: Yang
authors.affiliation: Department of Endocrine and Breast Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. Electronic address: tugang68@126.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Tu
mesh_terms: D052117:Benzodioxoles / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D000072645:Cancer-Associated Fibroblasts / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D011804:Quinolines / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C568862:4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta(c)quinoline; D052117:Benzodioxoles; D004967:Estrogens; D011804:Quinolines; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D004958:Estradiol; D002118:Calcium
keywords: Breast cancer; CAFs; Estrogenic effects; GPER; Tumor microenvironment