title: Inactivation of GPR30 reduces growth of triple-negative breast cancer cells: possible application in targeted therapy.
abstract: Triple-negative breast cancers lack estrogen receptor α (ERα), progesterone receptor, and do not overexpress human epidermal growth factor receptor 2 (Her-2). They are neither susceptible to endocrine therapy nor to a therapy using the anti-Her-2 antibody, trastuzumab. Therefore, an efficient targeted therapy is warranted. Triple-negative breast tumors frequently express membrane bound estrogen receptor G-protein coupled receptor (GPR30). As proof of principle, we analyzed the consequences of a knock-down of GPR30 expression on the growth regulation of triple-negative breast cancer cell lines. Cells of triple-negative breast cancer cell lines were transfected with siRNA against GPR30 or control siRNA, and cell growth was stimulated either with 10(⁻⁹) M 17β-estradiol or 10(⁻⁶) M 4-hydroxytamoxifen. Cell proliferation was measured using Alamar blue staining. Activation of c-Src and epidermal growth factor (EGF)-receptor was assessed using western blot. Expression of c-fos was quantified by reverse transcription polymerase chain reaction. Seven days after transfection with siRNA, GPR30 mRNA in triple-negative breast cancer cell lines MDA-MB-435 and HCC1806 was reduced by 74 and 90%, respectively. 10(⁻⁸) M 17β-estradiol enhanced proliferation of MDA-MB-435 to 129.6±5.4% of control (p<0.05) and HCC1806 to 156.9±15.4% of control (p<0.05), respectively. 10(⁻⁶) M 4-hydroxytamoxifen increased cell number of MDA-MB-435 to 121.0±6.9% of control (p<0.05) and HCC1806 to 124.5±12.1% of control (n.s.), respectively. This increased proliferation by the two estrogenic compounds was completely prevented by knock-down of GPR30 expression in both cell lines. In control cells, activity of Src kinase was increased 3-fold by estradiol and 3.8-fold using 4-hydroxytamoxifen. Transactivation of the EGF-receptor was similarly increased in both cell lines by 17β-estradiol and 4-hydroxytamoxifen. Both compounds increased c-fos expression 1.5- and 3.1-fold, respectively. Knock-down of GPR30 expression completely abolished activation of all these signaling pathways responsible for enhanced proliferation. A pharmacological inhibition of GPR30 by specific small molecular inhibitors might prove to be an appropriate targeted therapy of triple-negative breast cancer in the future.
authors.affiliation: Department of Obstetrics and Gynecology, Georg-August-University Göttingen, Göttingen, Germany. rainer.girgert@med.uni-guettingen.de
authors.forename: Rainer
authors.initials: R
authors.lastname: Girgert
authors.forename: Günter
authors.initials: G
authors.lastname: Emons
authors.forename: Carsten
authors.initials: C
authors.lastname: Gründker
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation; D004958:Estradiol / Q000494:pharmacology / Q000502:physiology; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D058990:Molecular Targeted Therapy; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D034622:RNA Interference; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000378:metabolism; D015398:Signal Transduction; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D018931:Antineoplastic Agents, Hormonal; D004958:Estradiol; D016760:Proto-Oncogene Proteins c-fos; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D011980:Receptors, Progesterone; D013629:Tamoxifen; C109584:GPER1 protein, human; C016601:afimoxifene; C508053:ERBB2 protein, human