The protein p22phox tightly interacts with NOX4 (1). resistance. Recent studies pointed to a key role of transforming growth factor (TGF-) in BRAFV600E-induced effects. The reactive oxygen species (ROS)-generating 5-hydroxytryptophan (5-HTP) NADPH oxidase NOX4, which is increased 5-hydroxytryptophan (5-HTP) in PTC, has been identified as a new key effector of TGF- in cancer, suggestive of a potential role in BRAFV600E-induced thyroid tumors. Here, using two human BRAFV600E-mutated thyroid cell lines and a rat thyroid cell line expressing BRAFV600E in a conditional manner, we show that NOX4 upregulation is controlled at the transcriptional level by the oncogene the TGF-/Smad3 signaling pathway. Importantly, treatment of cells with NOX4-targeted siRNA downregulates BRAFV600E-induced NIS repression. mutations are found in 40%C60% of the cases (7) and the most frequent is the point mutation that encodes the BRAFV600E oncogenic constitutively active protein kinase (50). This mutation is associated with more extensive disease, a higher rate of recurrence, and decreased survival (51). It is also associated with thyroid cell dedifferentiation with a decreased expression of thyroid functional genes (7). Innovation Our results establish a link between BRAFV600E and NOX4, which is confirmed by a comparative analysis of NOX4 expression in human (TCGA) and mouse thyroid cancers. Remarkably, analysis of human and murine BRAFV600E-mutated thyroid tumors highlights that the level of NOX4 expression is inversely correlated to thyroid differentiation, suggesting that other genes involved in thyroid differentiation in addition to NIS might be silenced by a mechanism controlled by NOX4-derived ROS. The property of thyroid cells to accumulate iodide is mediated by the sodium/iodide symporter (NIS) (13). This is clinically highly relevant because it enables treatment of thyroid cancers with radioiodine. However, BRAFV600E-positive tumors are often associated with a significant decrease or a complete loss of NIS expression. BRAF mediates the signal transduction of the mitogen-activated protein kinase (MEK)-extracellular-signal regulated kinase (ERK) pathway and the presence of a BRAF mutation predicts for a high sensitivity to MEK inhibitors in human cancers (28). Thus, BRAFV600E-positive thyroid cancer cell lines are sensitive to the growth suppressive effects of mitogen-activated protein kinase (MAPK) pathway inhibitors (28, 38) and small-molecule BRAF or MEK inhibitors restore radioiodine incorporation in the thyroid tumors of transgenic mice with doxycycline-inducible expression of BRAFV600E (8). However, it has been also observed that MEK inhibition did not rescue BRAFV600E-induced functional NIS repression in a rat thyroid cell line (41) and in some patients harboring thyroid cancer with BRAF mutation indicating that the MAPK pathway inhibition did not always fully revert cancer progression and BRAFV600E-induced effects (19). A possible explanation is that MAPK signaling is incompletely inhibited in some BRAF-mutant tumors because of relief of negative feedback effects on upstream components of the pathway (30, 35). The strong activation of the MAPK pathway also activates additional pathway(s) contributing to dedifferentiation and epithelialCmesenchymal transition (EMT) in tumors harboring oncogenic BRAF. Several studies have shown an important role of transforming growth factor 1 5-hydroxytryptophan (5-HTP) (TGF-) in this process. TGF- is overexpressed in human tumors (40), including thyroid malignancies (31, 47), and is a potent pro-oncogenic and prometastatic factor. Its role in cancer biology and in cell signaling is complex, and the cellular context appears to be a crucial determinant of the ultimate outcome of TGF- signaling in normal and tumor cells. TGF- was first shown to play a major role as a local modulator of thyroid by inhibiting both growth and differentiation in several species (17, 44, 46). It Leuprorelin Acetate can be a potent repressor of the expression of thyroid-specific functional genes, such as the NIS, thyroglobulin (Tg), and thyroperoxydase (TPO) (11, 24, 37). BRAFV600E expression induces the production 5-hydroxytryptophan (5-HTP) of functional TGF-1, which leads to a TGF–driven autocrine loop that mediates, at least in part, the effects of the BRAFV600E oncoprotein, in particular the decreased expression of NIS (41) and the promotion of cell migration, invasiveness, and EMT (25). However, the molecular basis of the BRAFV600E-induced TGF-1-loop in the development of thyroid cancers remains unclear. Recent observations suggest that reactive oxygen species (ROS) play an important role in the TGF-1-induced EMT and cell.