Negative Regulation of Human Granulosa Tumor Cell Survival by TGF-Beta.

Abstract

Disruption to the regulatory pathways that govern granulosa cell proliferation, differentiation, and death during ovarian development and folliculogenesis can result in a variety of disorders, including granulosa cell tumor (GCT) formation. Candidate growth regulatory molecules produced within the ovary include several TGF-beta superfamily members, but the specific stimuli and intracellular signalling pathways which determine human granulosa cell proliferation and survival are still poorly understood. We have used a human ovarian cancer cell line of GCT origin, the KGN cells, to study TGF-beta superfamily actions. We have recently reported that this GCT line exhibited very little expression of the TGF-beta accessory receptor betaglycan, a TGF-beta and inhibin co-receptor which determines cellular sensitivity to these ligands. We hypothesized that GCTs would be refractory to the growth-inhibitory effects of TGF-beta and/or inhibin and that overexpression of betaglycan would restore responsiveness to TGF-beta mediated growth inhibition. To test this hypothesis, KGN cells were stably transfected with a wildtype betaglycan expression construct or the empty vector as a control. Overexpression of betaglycan in the KGN cells restored sensitivity to TGF-beta and inhibin in luciferase reporter assays but had little effect on cell proliferation, cell cycle progression, or death in either GCT cell line as determined by flow cytometry. Furthermore, treatment with either exogenous TGF-beta2 or inhibin A, INHA knockdown, or neutralisation of endogenous TGF-betas also did not significantly affect cell survival and proliferation. As the GCT cells are tumour cell lines which exhibit constitutively active NF-kappaB, which is a pro-survival factor, we pre-treated the cells with the chemical NF-kappaB inhibitor, BAY11-7082. Under these conditions, overexpression of betaglycan resulted in a decrease in proliferation (15%; p<0.05) and an increase in apoptosis (300% p<0.05). The addition of TGF-beta1 and TGF-beta2 dose-dependently decreased cell viability, and this effect was enhanced by the presence of betaglycan. However, knockdown of INHA expression did not affect cell viability even in the presence of BAY11-7082. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from betaglycan- and vector-transfected GCT cells. 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 overexpression of betaglycan in the GCT cells increased both the basal and TGF-beta2-induced levels of activated SMAD3, with a smaller effect observed on activated SMAD2. Collectively, these data establish the TGF-beta-SMAD2/3-betaglycan signalling pathway as an important regulator of granulosa cell viability and suggest that deficiency of this pathway and/or inhibition by NF-kappaB contributes to dysregulated granulosa cell growth and cancer. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds. (platform)