title: GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines.
abstract: BACKGROUND
  Gonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R) activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed.
  
  
  METHODS
  GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125I-ligand binding and stimulation of 3H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting.
  
  
  RESULTS
  GnRH-R immunoscoring was highest in hormone receptor (triple) negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231). After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation.
  
  
  CONCLUSIONS
  Breast cancers exhibit a range of GnRH-R immunostaining, with higher levels of expression found in triple-negative and grade 3 cancers. However, functional cell surface receptors are rare in cultured cells. Intense GnRH-R signaling in transfected breast cancer cells did not markedly inhibit growth, in contrast to transfected HEK 293 cells indicating the importance of intracellular context. GnRH-R signaling could not counteract IGF-I receptor-tyrosine kinase addiction in MCF-7 cells. These results suggest that combinatorial strategies with growth factor inhibitors will be needed to enhance GnRH anti-proliferative effects in breast cancer.
authors: Medical Research Council Human Reproductive Sciences Unit, The Queen's Medical Research Institute, Little France Crescent, Old Dalkeith Road, Edinburgh EH16 4TJ, UK. kevinmorganxhrsu@gmail.com
authors: Kevin
authors: K
authors: Morgan
authors: Colette
authors: C
authors: Meyer
authors: Nicola
authors: N
authors: Miller
authors: Andrew H
authors: AH
authors: Sims
authors: Ilgin
authors: I
authors: Cagnan
authors: Dana
authors: D
authors: Faratian
authors: David J
authors: DJ
authors: Harrison
authors: Robert P
authors: RP
authors: Millar
authors: Simon P
authors: SP
authors: Langdon
mesh_terms: D000704:Analysis of Variance; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D046228:Microarray Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D011966:Receptors, LHRH / Q000378:metabolism*; D014162:Transfection; D017329:Triptorelin Pamoate / Q000494:pharmacology
chemical_list: D018931:Antineoplastic Agents, Hormonal; D007295:Inositol Phosphates; D011966:Receptors, LHRH; D017329:Triptorelin Pamoate