Betaglycan Alters NFκB-TGFβ2 Cross Talk to Reduce Survival of Human Granulosa Tumor Cells

The molecular pathways controlling granulosa cell tumor (GCT) survival are poorly understood. In many cell types, nuclear factor-kappa B (NF kappa B) and TGF beta coordinately regulate cell survival to maintain tissue homeostasis. Because GCT cell lines exhibit constitutively activated NF kappa B, we hypothesized that NF kappa B blocks TGF beta-mediated cell death in GCT cells. To test this hypothesis, we used the human GCT cell line KGN, which exhibits loss of betaglycan, a TGF beta co-receptor. After inhibition of NF kappa B in KGN cells, re-expression of betaglycan resulted in a decrease in cell viability, which was further decreased by TGF beta 2. Intriguingly, TGF beta 2 increased NF kappa B reporter activity in control cells, but betaglycan expression suppressed both basal and TGF beta 2-stimulated NF kappa B activity. Chemical inhibition of Mothers against decapentaplegic homolog 2/3 (SMAD2/3) signaling or SMAD2/3 gene silencing revealed that both SMADs contributed to cell survival. Furthermore, inhibiting NF kappa B activity resulted in a specific reduction in SMAD3 expression. Conversely, overexpression of SMAD3 increased basal NF kappa B activity and countered betaglycan-mediated suppression of NF kappa B activity. Finally, ERK1/2 activation emerged as the point of convergence of NF kappa B, SMAD3, and TGF beta 2/betaglycan governance of GCT cell viability. Key findings in KGN cells were reproduced in a second GCT cell line, COV434. Collectively, our data establish that both SMAD2/3 and NF kappa B signaling pathways support GCT cell viability and suggest the existence of a positive feedback loop between NF kappa B and SMAD3 signaling in late-stage GCT. Furthermore, our data suggest that loss of betaglycan during tumor progression in GCT alters the functional outcomes generated by NF kappa B and TGF beta pathway cross talk. (Molecular Endocrinology 27: 466-479, 2013)