Granulin-Epithelin Precursor (GEP): A new molecular target in ovarian cancer (OVCA) growth factor signaling.
Authors: Mitchell R. Kamrava, Emilyn U. Alejandro, Chad M. Michener, Elizabeth Meltzer, Fiona Simpkins, Elise C. Kohn
Journal: Cancer Research (2004)

2421   Previously we identified the Granulin-Epithelin Precursor (GEP) protein as differentially expressed between invasive and low malignant potential ovarian tumors. GEP is a 68-88 kD glycosylated protein that has been shown to regulate cell proliferation, invasion, and to protect against anoikis. While the cellular effects of GEP are being elucidated, little is known about the effectors responsible for regulating its expression. Lysophosphatidic acid (LPA) and endothelin-1 (ET-1) are growth factors known to be growth and invasion stimulators in OVCA. Both are G-protein coupled receptor (GPCR) ligands that signal through many pathways including cAMP. We hypothesized that GEP may be induced by LPA and ET-1 and that these growth factor signals may be amplified through the up-regulation of GEP. HEY-8 and OVCAR3 human OVCA cell lines were treated with these agents and modulators of the cAMP and MAPK pathways to map the events involved in GEP production. Cellular lysates and conditioned medium were collected and GEP protein expression levels were assessed by immunoprecipitation and Western blot analysis with confirmation by Northern analysis. Cell proliferation studies were done using a MTT assay. ET-1 and LPA increase GEP protein expression at concentrations seen physiologically in the blood and ascites of OVCA patients with maximal stimulation at 50nM and 20uM, respectively. Stimulants of the cAMP pathway, pertussis toxin, cholera toxin, and forskolin, increased GEP production in a dose-dependent manner with maximal response at 0.5 ug/ml, 0.5 ug/ml and 13 uM, respectively. cAMP propagates messages through protein kinase A (PKA) and through a less well characterized pathway, EPAC/Rap-1, a guanine-nucleotide-exchange factor and its small G-protein to activates MAP kinases downstream. Increasing H89 concentrations (500nm-10uM), a PKA inhibitor, does not decrease GEP production in HEY-8 cells and in fact has a stimulatory effect. However, 8-CTP-2-O-Me-cAMP, a selective activator of the EPAC/Rap-1 pathway induced GEP production in a dose dependent fashion with maximal stimulation at 300uM. OVCAR3 cells had both the PKA and EPAC pathways active, with the EPAC pathway predominating. This explains the stimulation by H89 as a shunting of cAMP stimuli through the EPAC/Rap-1 pathway. All stimulatory responses through the EPAC pathway, from ET-1 to CTP-c-AMP, were abrogated by pretreatment of cells with PD98059 at 75 uM, a MEK-1 inhibitor. Inhibition of GEP using a neutralizing antibody reduced the proliferative drive of forskolin, CTP-c-AMP, and ET-1. These results demonstrate that ET-1 stimulates GEP in HEY-8 and OVCAR3 by signaling through the GPCR family predominantly down the cAMP/EPAC/MAPK pathway. GEP is also a component of the ET-1 and LPA growth factor signal amplification pathway driving OVCA cell proliferation. Thus, GEP and its stimulation pathway is an important new molecular target in OVCA.