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 IGFI 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 rephosphorylation. 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 receptortyrosine 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