The function of the NAD(P)H oxidases (NOXs) family member NOX4 is to generate reactive oxygen species (ROS), however, the molecular function of NOX4 has not been fully studied and waiting to be clarified. To elucidate the function of endogenous Nox4 in human thyroid carcinomas, papillomatosis thyroid cancer cells were used to study the cell growth by knocking down the expression of NOX4 and knocking out its functional partner p22phox/CYBA. As a result, the increasement of mitochondrial ROS(mROS) was abolished due to both knockdown of NOX4 and p22phox knockout in hypoxia, which destabilized HIF1α decreasing glycolysis and retarded cell growth. These data suggests that Nox4 is potent oncotarget due to its role in regulating glycolysis through mROS-HIF1α pathway, thereby mediating proliferation in thyroid carcinomas.

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Advanced differentiated thyroid cancer cells are subjected to extreme nutritional starvation which contributes to develop resistance to treatments; however, the underlying mechanism remains unclear. Cells were subjected to serum deprivation by culture in medium containing 0.5% fetal bovine serum. A CCK8 assay, cell death Detection ELISAPLUS kit, and PI staining were conducted to determine cell viability, cell apoptosis, and cell cycle, respectively. NADPH oxidase 4 (NOX4) knockdown–stable cell lines were generated by lentivirus-mediated shRNA knockdown in BCPAP cells and TPC-1 cells. Etoposide and doxorubicin, two chemotherapeutic drugs, as well as lenvatinib were utilized to determine the effect of NOX4 on drug resistance. Lenvatinib-resistant BCPAP cells (LRBCs) were established to confirm this effect. The underlining mechanisms of NOX4 under starvation were explored using western blot. Finally, GLX351322, an inhibitor targeting NOX4, was used to inhibit NOX4-derived ROS in vitro and detect its effect on drug resistance of tumor cells in vivo. NOX4 is overexpressed under serum deprivation in BCPAP or TPC-1 cells. NOX4 knockdown impairs cell viability, increases cell apoptosis, extends G1 phase during cell cycle and modulates the level of energy-associated metabolites in starved cells. When the starved cells or LRBCs are treated with chemotherapeutic drugs or Lenvatinib, NOX4 knockdown inhibits cell viability and aggravates cell apoptosis depending on NOX4-derived ROS production. Mechanistically, starvation activates TGFβ1/SMAD3 signal, which mediates NOX4 upregulation. The upregulated NOX4 then triggers ERKs and PI3K/AKT pathway to influence cell apoptosis. GLX351322, a NOX4-derived ROS inhibitor, has an inhibitory effect on cell growth in vitro and the growth of BCPAP-derived even LRBCs-derived xenografts in vivo. These findings highlight NOX4 and NOX4-derived ROS as a potential therapeutic target in resistance to PTC.