Pancreatic adenocarcinoma upregulated factor (PAUF) was initially identified as a secreted protein that is substantially expressed in pancreatic ductal adenocarcinoma (PDAC). PAUF also affects invasiveness, motility, and the proliferation of cells in several types of cancer. Recently, PAUF was reported to play a pivotal role in the TLR4-mediated migration and invasion of PDAC cells. However, the mechanism inducing PAUF expression and its functional role in TGF-β-stimulated PDAC cells have not yet been studied. Thus, we first assessed whether TGF-β regulates PAUF expression in several PDAC cell lines and found a significant increase in PAUF expression in Smad signaling-positive Panc-1 cells treated with TGF-β. We also found that the PAUF promoter region contains a Smad-binding element. TGF-β-treated Panc-1 cells showed an increase in PAUF promoter activity, but this effect was not observed in TGF-β-stimulated Smad4-null BxPC-3 cells. Restoring Smad4 expression increased the PAUF promoter activity and expression in Smad4-overexpressing BxPC-3 cells treated with TGF-β. We further found that PAUF aggravated the TGF-β-induced epithelial–mesenchymal transition (EMT) in Panc-1 and BxPC-3 cells via the activation of MEK-ERK signaling. These results indicate that TGF-β/Smad signaling-mediated upregulation of PAUF plays a crucial role in EMT progression by activating the TGF-β-mediated MEK-ERK signaling pathway.

Pancreatic adenocarcinoma upregulated factor (PAUF), a novel secreted protein highly expressed in pancreatic ductal adenocarcinoma, also influences cell invasiveness, motility, and proliferation in several cancer types. Transforming growth factor-beta (TGF-β)-induced PAUF expression enhances cancer cell migration and invasion in pancreatic ductal adenocarcinoma through mitogen-activated protein kinase (MEK)–extracellular signal-regulated kinase (ERK) activation; however, the roles of PAUF in regulating epithelial–mesenchymal transition (EMT) and promoting lung metastasis in hepatocellular carcinoma (HCC) remain unclear. Thus, we investigated the regulatory mechanisms and functional roles of TGF-β-induced PAUF expression in the HCC cell lines HepG2 and Huh-7, which showed high and low expression of intact TGF-β type I and II receptors, respectively. We found that TGF-β-induced PAUF expression is mediated through the activation of the TGF-β type I/II receptor–Smads signaling pathway and that PAUF promotes EMT-associated migration and invasion by stimulating the MEK–ERK signaling cascade. In vivo studies further demonstrated that PAUF plays a critical role in lung metastatic potential, as PAUF knockdown HepG2 cells exhibited markedly reduced pulmonary metastasis, whereas PAUF-overexpressing Huh-7 cells showed substantially enhanced lung metastasis. This study identifies PAUF as a critical promoter of lung metastatic potential in HCC cells and a potential therapeutic target for HCC.