Betaglycan as a Determinant of TGF-beta Superfamily Action in Human Granulosa Tumor Cells.

Abstract

Granulosa cell tumors (GCTs) of the ovary belong to a group of ovarian sex cord stromal tumors that account for approximately 5% of total ovarian cancer incidences. The mechanisms by which ovarian granulosa cells undergo malignant transformation are poorly understood but are likely to involve the disruption of regulatory pathways that function during normal folliculogenesis. The type III TGF-beta receptor, betaglycan, and its ligands are essential regulatory factors in reproduction. Within the ovary, these factors regulate multiple processes in normal granulosa cell biology in a species-dependent manner. We have recently reported that human GCTs exhibit low betaglycan expression relative to the normal pre-menopausal ovary and that reduced betaglycan expression may contribute to the malignant behavior of granulosa tumor cells, increasing their ability to migrate, invade extracellular matrix, and metastasize. In two human GCT cell lines, the COV434 and KGN cells, we demonstrated that INHA gene silencing blocked the betaglycan-mediated inhibition of migration and invasion whereas both INHA gene silencing and TGF-beta neutralization abolished the betaglycan-mediated increase in adhesion to substrate. The objective of the current study was to determine which signaling pathways are modulated by the presence of betaglycan on GCT cells. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from KGN and COV434 cells which had been stably transfected with a full-length wildtype betaglycan or a vector control. The relative levels of activation of specific pathways were determined by western blot analyses using activation-specific antibodies against SMAD2, SMAD3, and SMAD1/5. This analysis revealed that the COV434 and KGN cells exhibit different profiles of activation of each of the SMAD molecules, with COV434 cells exhibiting lower basal and stimulated levels of SMAD2, SMAD3, and SMAD1/5 than KGN cells. The presence of betaglycan did not significantly affect basal SMAD activation, but in both COV434 and KGN cells, the expression of betaglycan significantly increased the activation of SMAD2 in response to TGF-beta2. Furthermore, the knockdown of INHA expression further enhanced TGF-beta2 mediated SMAD2 activation in the betaglycan-expressing KGN cells. Collectively, our data indicate that the presence of betaglycan on GCT cells impacts on both inhibin- and TGF-beta-mediated GCT behaviors. Furthermore, the elimination of autocrine inhibin actions in the GCT cells enhanced TGF-beta2 stimulated SMAD2 activation in a betaglycan-dependent manner, suggesting that the presence of betaglycan on the surface of GCT cells counterbalances inhibin- and TGF-beta mediated signalling. (poster)