Signaling and Antiproliferative Effects Mediated by GnRH Receptors After Expression in Breast Cancer Cells Using Recombinant Adenovirus

Abstract

GnRH receptors (GnRH-Rs) are found in human cancers, including those of the breast, and GnRH can inhibit the growth of cell lines derived from such cancers. Although pituitary and extrapituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extrapituitary GnRH-Rs have low affinity for GnRH analogs and may not activate PLC or discriminate between agonists and antagonists in the same way as pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in breast cancer cells differ from those in gonadotropes. We found no evidence for endogenous GnRH-Rs in MCF7 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at a multiplicity of infection of 10 or greater, at least 80% expressed GnRH-Rs. These had high affinity (K(d) for [(125)I]buserelin, 1.4 nM) and specificity (rank order of potency, buserelin>GnRH>>chicken GnRH-II) and mediated stimulation of [(3)H]IP accumulation. Increasing viral titer [from multiplicity of infection, 3-300] increased receptor number (10,000-225,000 sites/cell) and [(3)H]IP responses. GnRH stimulated ERK2 phosphorylation in Ad GnRH-R-infected cells, and this effect, like stimulation of [(3)H]IP accumulation, was blocked by GnRH-R antagonists. GnRH also inhibited [(3)H]thymidine incorporation into Ad GnRH-R-infected cells (but not control cells). This effect was mimicked by agonist analogs and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at density comparable to that in gonadotropes, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotropes, and increasing expression of high affinity GnRH-Rs can dramatically enhance the direct antiproliferative effect of GnRH agonists on breast cancer cells.

Gonadotropes and Gonadotropin-Releasing Hormone Signaling

“…208,216,219,220 The simplest explanation for differing pharmacology with pituitary and extrapituitary GnRHRs would be that they are mediated by distinct receptors, but the type I hGnRHR transcripts expressed in pituitary and extrapituitary sites are identical. 213,218,223 Such experiments with heterologous GnRHRs are clearly consistent, with a role for GnRHR in the mediation of direct effects on cell fate. However, humans express a type II GnRHR pseudogene that does not encode functional GnRHRs.…”

“…208,209,211,215,216,219,236 A further distinction is that ligands acting as competitive antagonists of pituitary GnRHRs can sometimes mimic the effects of agonists in cancer cells, implying that the agonist-antagonist dichotomy established for pituitary GnRHRs may not be applicable to extrapituitary GnRHRs. 213,218,223 However, when recombinant adenovirus was used to express type I GnRHRs in these cells (at levels similar to those found in gonadotropes), the heterologously expressed receptors were very similar (in terms of binding affinity, ligand specificity, and G q/11 coupling) to those of the native GnRHRs in gonadotropes. 642 The cloning of type II GnRHRs in primates 79 and the demonstration that GnRH II has potent antiproliferative and/or apoptotic effects 217,221,224,225,228,234 raised the possibility that the functional differences reflected mediation by type I and type II receptors.…”

Retracted Article : Gonadotropin releasing hormone analogue (GnRHa) alters the expression and activation of Smad in human endometrial epithelial and stromal cells

“…Human endometrium expresses both forms of GnRH receptors, however it remains to be determined whether GnRH and Antide interact with one or both GnRH receptors, or Antide could convert an antagonist action into an agonist as seen in other cell types [51,52]. Both GnRH receptors are reported to mediate antiproliferative and apoptotic effects of GnRH [13,53]. …”

Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans

“…In addition, the receptor internalizes slowly via clathrin-coated vesicles, and this process occurs independently of ␤-arrestin and dynamin (226, 229 -231). The unusual resistance of the mammalian GnRH receptor to desensitization may be essential for mediating its direct antiproliferative effect (will be discussed in detail below), which requires sustained ligand stimulation and is shown to be ineffective by receptors having a carboxyl-terminal tail (230,232).…”