Nox4-derived ROS Signaling Contributes to TGF-β-induced Epithelial-mesenchymal Transition in Pancreatic Cancer Cells

Transforming growth factor (TGF)-β induces epithelial-mesenchymal transition (EMT) in pancreatic adenocarcinoma. In this study, we investigated how NADPH oxidase (Nox) 4-generated reactive oxygen species (ROS) regulate TGF-β-induced EMT in pancreatic cancer cells. Materials and Methods: Pancreatic cancer cells were transfected with Nox4 siRNAs or PTP1B mutants and subjected to TGF-β-induced EMT assay. Expression of Nox4, TGF-β, and N-cadherin was immunohistochemically-examined with patient tumor samples. Results: Treatment of pancreatic cancer cells with TGF-β induced Nox4 expression, indicating that Nox4 represents a major source for ROS production. The Nox4 inhibitor diphenylene iodonium and Nox4 siRNAs blocked TGF-β-induced EMT phenotype including morphological changes, augmented migration, and altered expression of E- cadherin and Snail. Furthermore, PTP1B as a redox-sensor for Nox4-derived ROS participated in TGF-β-promoted EMT. Nox4, TGF-β, and N-cadherin were up-regulated in tumors from pancreatic cancer patients. Conclusions: These findings suggest that Nox4-derived ROS, at least in part, transmit TGF- β-triggered EMT signals through PTP1B in pancreatic cancer. Epithelial-mesenchymal transition (EMT) is the process that converts polarized epithelial cells, tightly bound to each other, into fibroblastic cells with migratory potential (1). During EMT, epithelial cells undergo numerous biological and biochemical changes in order to acquire a fibroblast-like phenotype. These include acquisition of both spindle-like morphology and cell-to-cell dissociation, up-regulation of mesenchymal markers (N-cadherin and vimentin), down- regulation of epithelial markers (E-cadherin and ZO-1), and induction of specific transcription factors (Snail and Twist) (2). EMT is vital for morphogenesis during embryonic development and is also implicated in tumor cell invasion and migration. EMT associated with cancer is promoted by growth factors, cytokines, and matrix proteins secreted in tumor microenvironments. Pancreatic cancer, which is characterized by strong chemoresistance and high metastatic potential, is one of the most deadly cancer types. The critical role of EMT has been implicated in aggressive malignancy and lethality of pancreatic carcinoma (3). In pancreatic adenocarcinoma, TGF-β is the major inducer of EMT, which is a prerequisite for tumor invasiveness and metastasis (4), and overexpression of TGF-β