Abstract
BackgroundGonadotrophin 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.MethodsGnRH-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.ResultsGnRH-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.ConclusionsBreast 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
Highlights
Gonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients
The ability of GnRH agonist to inhibit cell growth appears to correlate with the level of GnRH receptor expression at the cell surface and with the magnitude of inositol phosphate production elicited by receptor activation [8,9]
GnRH receptor activation coupled to Gaq/11-Gbg proteins leads to elevation of intracellular Ca2+ levels, altered cytoskeletal function and changes in protein kinase activity, including protein kinase C (PKC), mitogen activated serine/ threonine kinases (MAPkinases, MAPK) and stress-activated kinases Cell-type specific effects of GnRH receptor activation on levels of phosphorylated-ERK1/2 (p-ERK1/ 2) have been observed [8,9] which probably reflect the complexity of protein scaffolds interacting with and influencing MAPK
Summary
Gonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients. Endocrine suppression using gonadotropin releasing hormone (GnRH) analogs such as goserelin (a superagonist) is commonly used for the treatment of premenopausal estrogen-responsive breast cancer because it lowers plasma levels of estrogen by inhibiting secretion of luteinizing hormone and follicle stimulating hormone from the pituitary gland [1,2] and thereby slows estrogen-driven tumor growth. It has been speculated since a proportion of cancer cells express GnRH receptor, that activation or inhibition of GnRH receptor signaling may directly affect cell growth [3,4,5]. Effects of GnRH receptor signaling on transcription factor activity and gene expression downstream from MAPK are likely
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