NADPH Oxidase NOX4 Is a Critical Mediator of BRAFV600E-Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas.

AIMS: The BRAFV600E oncogene, reported in 40%-60% of papillary thyroid cancer (PTC), has an important role in the pathogenesis of PTC. It is associated with the loss of thyroid iodide-metabolizing genes, such as sodium/iodide symporter (NIS), and therefore with radioiodine refractoriness. Inhibition of mitogen-activated protein kinase (MAPK) pathway, constitutively activated by BRAFV600E, is not always efficient in resistant tumors suggesting that other compensatory mechanisms contribute to a BRAFV600E adaptive resistance. Recent studies pointed to a key role of transforming growth factor ß (TGF-ß) in BRAFV600E-induced effects. The reactive oxygen species (ROS)-generating NADPH oxidase NOX4, which is increased in PTC, has been identified as a new key effector of TGF-ß in cancer, suggestive of a potential role in BRAFV600E-induced thyroid tumors. RESULTS: 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 via the TGF-ß/Smad3 signaling pathway. Importantly, treatment of cells with NOX4-targeted siRNA downregulates BRAFV600E-induced NIS repression. Innovation and Conclusion: 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. This study opens a new opportunity to optimize thyroid cancer therapy. Antioxid. Redox Signal. 26, 864-877.

Redox homeostasis of breast cancer lineages contributes to differential cell death response to exogenous hydrogen peroxide.

AIMS: Cancer cells produce higher amounts of reactive oxygen species (ROS) than their normal counterparts. It has been suggested that a further increase in ROS concentration in these cells would lead to oxidative damage-driven death. Thus, we aimed to understand how the intra- and extracellular redox homeostasis differences set cell death response to ROS in breast cancer cell lines. MAIN METHODS: Intra- and extracellular ROS generation was evaluated in tumoral (MCF-7 and MDA-MB-231) and non-tumoral (MCF10A) breast epithelial cells, as well as H2O2 concentration in the culture medium, glutathione peroxidase (GPx), total superoxide dismutase (SOD) and catalase activities, extracellular H2O2 scavenging capacity and total thiol content. Cell viability was determined after H2O2 exposure using the MTT assay. KEY FINDINGS: We have found an increased extracellular ROS production in tumor cells when compared to the non-tumoral lineage. MCF10A cells had higher H2O2 concentration in the extracellular medium. Moreover, extracellular H2O2-scavenging activity was higher in MDA-MB-231 when compared to MCF10A and MCF-7. Regarding intracellular antioxidant activity, a lower GPx activity in tumor cell lines and a higher catalase activity in MDA-MB-231 were observed. Thiol content was lower in MDA-MB-231. Additionally, tumor cell lines were more sensitive to H2O2 exposure than the non-tumoral cells. SIGNIFICANCE: The present report shows that the capability to generate and metabolize ROS differ greatly among the breast cancer cell lines, thus suggesting that redox balance is finely regulated during carcinogenesis. Therefore, our data suggest that therapeutic approaches targeting the redox status might be useful in the treatment of breast tumors.

5'-AMP-Activated Protein Kinase Regulates Papillary (TPC-1 and BCPAP) Thyroid Cancer Cell Survival, Migration, Invasion, and Epithelial-to-Mesenchymal Transition.

BACKGROUND: Differentiated thyroid carcinomas (DTC) are associated with a good prognosis and a high survival rate. However, tumor recurrence occurs in approximately 20-30% of DTC patients, reinforcing the importance of identifying new molecular targets for cancer management. It has been shown that the 5'-AMP-activated protein kinase (AMPK) is over-activated in papillary thyroid cancer (PTC). This study aimed to investigate the effects of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an AMPK activator, on various aspects of thyroid cancer cell behavior, including cell survival, apoptosis, migration, invasion, and epithelial-to-mesenchymal transition (EMT), in the human thyroid cancer cell lines BCPAP and TPC-1. METHODS: BCPAP and TPC-1 cells were cultivated in Dulbecco's modified Eagle's medium, and the non-tumor-derived cell line Nthy-ORI was grown in RPMI. Cells were treated or not with AICAR for different periods of time. The cell growth rate, cell cycle phase, apoptosis, cell migration, and invasion were analyzed using transwell inserts, and EMT was quantified by the expression of mesenchymal and epithelial markers. RESULTS: AMPK is activated in thyroid cancer cell lines, and AICAR treatment further increased AMPK phosphorylation. After 48 hours of AICAR treatment, the percentage of cells in the G2/M phase decreased, and a G0/G1-phase arrest was induced in both cell lines. AMPK activation effectively induced apoptosis in the BCPAP and TPC-1 cancer cell lines, while no apoptosis induction was observed in Nthy-ORI cells. AICAR also reduced the migration of Nthy-ORI and BCPAP cells by 30% and approximately 60% in TPC-1 cells. AICAR had no effect on cell invasion in Nthy-ORI and TPC-1 cells, but a significant reduction of cell invasion was observed in BCPAP cells. AICAR induced a significant reduction of N-cadherin and no changes in the expression of vimentin or TCF/Zeb1 protein in BCPAP cells. No differences in the expression of EMT markers were found in the AICAR-treated Nthy-ORI cells. A remarkable reduction of vimentin, TCF/Zeb1, and N-cadherin protein expression was detected in the TPC-1 cells. CONCLUSIONS: Increased activation of AMPK in PTC cell lines leads to a strong antitumor response, as measured by the inhibition of cell proliferation, cell migration, and induction of cell death. AMPK activation also reverses EMT in TPC-1 cells.

A novel role for AMP-kinase in the regulation of the Na+/I--symporter and iodide uptake in the rat thyroid gland.

The aim of this study was to investigate the role of AMP-kinase (AMPK) in the regulation of iodide uptake by the thyroid gland. Iodide uptake was assessed in PCCL3 follicular thyroid cells exposed to the AMPK agonist 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), and also in rat thyroid glands 24 h after a single intraperitoneal injection of AICAR. In PCCL3 cells, AICAR-induced AMPK and acetyl-CoA carboxylase (ACC) phosphorylation decreased iodide uptake in a concentration-dependent manner, while the AMPK inhibitor compound C prevented this effect. In the thyroid gland of rats injected with AICAR, AMPK and ACC phosphorylation was increased and iodide uptake was reduced by ~35%. Under conditions of increased AMPK phosphorylation/activation such as TSH deprivation or AICAR treatment, significant reductions in cellular Na(+)/I(-)-symporter (NIS) protein (~41%) and mRNA content (~65%) were observed. The transcriptional (actinomycin D) and translational (cycloheximide) inhibitors, as well as the AMPK inhibitor compound C prevented AICAR-induced reduction of NIS protein content in PCCL3 cells. The presence of TSH in the culture medium reduced AMPK phosphorylation in PCCL3 cells, while inhibition of protein kinase A (PKA) with H89 prevented this effect. Conversely, the adenylyl cyclase activator forskolin abolished the AMPK phosphorylation response induced by TSH withdrawal in PCCL3 cells. These findings demonstrate that TSH suppresses AMPK phosphorylation/activation in a cAMP-PKA-dependent manner. In summary, we provide novel evidence that AMPK is involved in the physiological regulation of iodide uptake, which is an essential step for the formation of thyroid hormones as well as for the regulation of thyroid function.