title: Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231.
abstract: BACKGROUND
  Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion.
  
  
  METHODS
  Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay.
  
  
  RESULTS
  Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers.
  
  
  CONCLUSIONS
  We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.
authors.affiliation: Centro de Investigación Biomédica del Sur (CIBIS), Instituto Mexicano del Seguro Social (IMSS), Argentina No, 1, Col, Centro, 62790, Xochitepec, Morelos, Mexico. a_aguilar@unam.mx
authors.forename: Arturo
authors.initials: A
authors.lastname: Aguilar-Rojas
authors.forename: Maira
authors.initials: M
authors.lastname: Huerta-Reyes
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Maya-Núñez
authors.forename: Fabián
authors.initials: F
authors.lastname: Arechavaleta-Velásco
authors.forename: P Michael
authors.initials: PM
authors.lastname: Conn
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ulloa-Aguirre
authors.forename: Jesús
authors.initials: J
authors.lastname: Valdés
mesh_terms: D000199:Actins / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002064:Buserelin / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005434:Flow Cytometry; D005470:Fluorometry; D022001:Focal Adhesions / Q000187:drug effects; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D061986:MCF-7 Cells; D018613:Microscopy, Confocal; D009154:Mutation; D009361:Neoplasm Invasiveness; D058105:Polymerization / Q000187:drug effects; D011966:Receptors, LHRH / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D022502:Stress Fibers / Q000378:metabolism; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; C484844:GNRHR protein, human; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D020742:rhoA GTP-Binding Protein; D002064:Buserelin