Transcriptional regulation of human mucin gene MUC4 by TGF-β and EGF/TGF-α in pancreatic cancer cells involves a complex cross-talk between the Smads, MAPK, P13K, and PKA signaling pathways

Abstract

Human MUC4 mucin gene encodes a large transmembrane mucin that is overexpressed in human pancreatic carcinomas and most likely involved in carcinogenesis. TGF-13, which signals through the Smads proteins, is another key actor in pancreas cancer since Smad4 is inactivated in 50 % of the tumors. Our aim was to study MUC4 regulation by TGF-13 and EGF/TGF-a in pancreatic cancer cell lines CAPAN-1 (MUC4 + +), CAPAN-2 (MUC4 + ) and PANC-1 (MUC4 -) that bear different phenotypic and genotypic features. By transient transfections and gebshifi assays, we have identified eleven active Smad4 cis-elements within MUC4 promoter. By using a Stand4-/cell line and by overexpressing Smad2, Smad3 and Stand4 transcription factors in co-transfeetion experiments, we demonstrate that Smad2 and Stand4 directly up-regulate MUC4 promoter activity whereas Smad3 acts as a inhibitor of Smad4. Finally, RT-PCR and functional analyses indicate that (i) TGF-f3 treatment induces MUC4 transcription regardless of Stand4 functionality and (ii) a strong synergistic effect occurs between TGF-J3 and two growth factors of the EGF family, EGF and TGF-a, which suggests that MAPK signaling pathway is also involved. Using specific pharmacological inhibitors, we indeed confirmed that MAPK is involved in the TGF-J3and TGF-c~-mediated up-regnlation of MUC4 promoter but that PI3K and PKA kmases are also playing a nonnegligible role. In conclusion, our results demonstrate that MUC4 transcriptional regulation by TGF-[3 in pancreatic cancer ceils is complex and is the result of a cross-talk between the Smads, MAPK, PI3K and PKA signaling cascades. MUC4 appears thus as a potent target gene of growth factors known to be involved in cell proliferation and differentiation in pancreatic cancer.