title: Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER.
abstract: BACKGROUND
  Estrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear.
  
  
  METHODS
  MCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation.
  
  
  RESULTS
  In the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions.
  
  
  CONCLUSIONS
  Our results suggest that non-genomic signaling by GPER contributes, at least in part, to the survival of breast cancer cells, particularly in the presence of ER-targeted therapies involving SERMs and SERDs. Our results further suggest that GPER expression and FOXO3a localization could be utilized as prognostic markers in breast cancer therapy and that GPER antagonists could promote apoptosis in GPER-positive breast cancers, particularly in combination with chemotherapeutic and ER-targeted drugs, by antagonizing estrogen-mediated FOXO3a inactivation.
authors.affiliation: Department of Internal Medicine and UNM Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA. e.zekas@gmail.com.
authors.forename: Erin
authors.initials: E
authors.lastname: Zekas
authors.affiliation: Department of Internal Medicine and UNM Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA. eprossnitz@salud.unm.edu.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Prossnitz
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000008:administration & dosage / Q000378:metabolism*; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D034741:RNA, Small Interfering; D020849:Raloxifene Hydrochloride / Q000008:administration & dosage; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D013629:Tamoxifen / Q000008:administration & dosage
chemical_list: D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C109584:GPER1 protein, human; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D013629:Tamoxifen; D020849:Raloxifene Hydrochloride