title: 17β-estradiol-induced growth of triple-negative breast cancer cells is prevented by the reduction of GPER expression after treatment with gefitinib.
abstract: Triple-negative breast cancers (TNBCs) are neither susceptible to endocrine therapy due to a lack of estrogen receptor α expression nor trastuzumab. TNBCs frequently overexpress epidermal growth factor receptor (EGFR) and membrane bound estrogen receptor, GPER. To a certain extent the growth of TNBCs is stimulated by 17β-estradiol via GPER. We analyzed whether inhibition of EGFR by gefitinib reduces the expression of GPER and subsequent signal transduction in TNBC cells. Dependence of proliferation on 17β-estradiol was determined using Alamar Blue assay. Expression of GPR30 and activation of c-src, EGFR and cAMP-responsive element binding (CREB) protein by 17β-estradiol was analyzed by western blotting. Expression of c-fos, cyclin D1 and aromatase was determined using RT-PCR. Gefitinib reduced GPER expression concentration‑ and time‑dependently. In HCC70 cells, GPER expression was reduced to 15±11% (p<0.05) after treatment with 200 nM gefitinib for four days, and in HCC1806 cells GPER expression was reduced to 39±5% (p<0.01) of the control. 17β-estradiol significantly increased the percentage of HCC1806 cells within 7 days to 145±29% of the control (HCC70, 110±8%). This increase in cell growth was completely prevented in both TNBC cell lines after GPR30 expression was downregulated by treatment with 200 nM gefitinib. In HCC1806 cells, activation of c-src was increased by 17β-estradiol to 350±50% (p<0.01), and gefitinib reduced src activation to 110%. Similar results were obtained in the HCC70 cells. Phosphorylation of EGFR increased to 240±40% (p<0.05) in the HCC1806 cells treated with 17β-estradiol (HCC70, 147±25%). Gefitinib completely prevented this activation. Phosphorylation of CREB and induction of c-fos, cyclin D1 and aromatase expression by 17β-estradiol were all prevented by gefitinib. These experiments conclusively show that reduction of GPER expression is a promising therapeutic approach for TNBC.
authors.affiliation: Department of Gynecology and Obstetrics, School of Medicine, University of Göttingen, D-37075 Göttingen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Girgert
authors.affiliation: Department of Gynecology and Obstetrics, School of Medicine, University of Göttingen, D-37075 Göttingen, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Emons
authors.affiliation: Department of Gynecology and Obstetrics, School of Medicine, University of Göttingen, D-37075 Göttingen, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Gründker
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001141:Aromatase / Q000378:metabolism; D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019938:Cyclin D1 / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016762:Genes, fos; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology*; D011960:Receptors, Estrogen / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C530104:CCND1 protein, human; C109584:GPER1 protein, human; D011799:Quinazolines; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D019938:Cyclin D1; D004958:Estradiol; D001141:Aromatase; C561410:CYP19A1 protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D000077156:Gefitinib