Betaglycan alters NF{kappa}B-TGF{beta}2 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-kappaB (NFkappaB) and TGFbeta coordinately regulate cell survival to maintain tissue homeostasis. Because GCT cell lines exhibit constitutively activated NFkappaB, we hypothesized that NFkappaB blocks TGFbeta-mediated cell death in GCT cells. To test this hypothesis, we used the human GCT cell line KGN, which exhibits loss of betaglycan, a TGFbeta co-receptor. After inhibition of NFkappaB in KGN cells, re-expression of betaglycan resulted in a decrease in cell viability, which was further decreased by TGFbeta2. Intriguingly, TGFbeta2 increased NFkappaB reporter activity in control cells, but betaglycan expression suppressed both basal and TGFbeta2-stimulated NFkappaB 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 NFkappaB activity resulted in a specific reduction in SMAD3 expression. Conversely, overexpression of SMAD3 increased basal NFkappaB activity and countered betaglycan-mediated suppression of NFkappaB activity. Finally, ERK1/2 activation emerged as the point of convergence of NFkappaB, SMAD3, and TGFbeta2/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 NFkappaB signaling pathways support GCT cell viability and suggest the existence of a positive feedback loop between NFkappaB 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 NFkappaB and TGFbeta pathway cross talk.