Correlation of glypican-1 expression with TGF-β, BMP, and activin receptors in pancreatic ductal adenocarcinoma

Abstract

Glypican1 (GPC1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors as well as for members of the TGF-β family. GPC1 plays a role in pancreatic cancer by regulating growth factor responsiveness. In view of the importance of members of the TGF-β family in pancreatic cancer, in the present study, the role of GPC1 in TGF-β, BMP and activin signaling was analyzed. Quantitative RT-PCR and immunohistochemistry were utilized to analyze GPC1 and TGF-β, BMP and activin receptor expression levels. Panc-1 and T3M4 pancreatic cancer cells were transfected in a stable manner with a GPC1 antisense expression construct. Anchorage-dependent and -independent growth was determined by MTT and soft agar assays. TGF-β1, activin-A and BMP-2 responsiveness was determined by MTT assays and immunoblotting with p21, p-Smad1, and p-Smad2 antibodies. QRT-PCR demonstrated increased GPC1 mRNA levels in pancreatic ductal adenocarcinoma (PDAC) compared to normal pancreatic tissues (NPT), as described previously. There was a significant correlation between GPC1 mRNA levels and TβRII, act-R1a, act-R1b, act-R2a, BMP-R1a, and BMP-R2 mRNA expression in NPT. In contrast, GPC1 mRNA expression correlated directly with act-R1a and BMP-R1a in N0 PDAC cases and with act-R2a and BMP-R1a in lymph node positive cases. Down-regulation of GPC1 resulted in increased doubling time in Panc-1 but not in T3M4 cells, and decreased anchorage-independent growth in both cell lines. GPC1 down-regulation resulted in a slightly altered response towards TGF-β1, activin-A and BMP-2 in terms of growth, p21 induction and Smad2 phosphorylation. In conclusion, enhanced GPC1 expression correlates with BMP and activin receptors in pancreatic cancer. GPC1 down-regulation suppresses pancreatic cancer cell growth and slightly modifies signaling of members of the TGF-β family of growth factors.