Aims: The BRAFV600E oncogene, reported in 40-60% of papillary thyroid carcinoma (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 radioiodide-refractoriness. Inhibition of 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 TGF-β in BRAFV600E-induced effects. The reactive oxygene species (ROS)-generating NADPH oxidase NOX4, which is increased in PTC, has been identified as a new key effector of TGF-β 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 up-regulation is controlled at the transcriptional level by the oncogene via the TGF-β/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

Radioiodine therapy (RAI) is a standard and effective therapeutic approach for differentiated thyroid cancers (DTCs) based on the unique capacity for iodide uptake and accumulation of the thyroid gland through the Na⁺/I⁻ symporter (NIS). However, around 5–15% of DTC patients may become refractory to radioiodine, which is associated with a worse prognosis. The loss of RAI avidity due to thyroid cancers is attributed to cell dedifferentiation, resulting in NIS repression by transcriptional and post-transcriptional mechanisms. Targeting the signaling pathways potentially involved in this process to induce de novo iodide uptake in refractory tumors is the rationale of “redifferentiation strategies”. Oxidative stress (OS) results from the imbalance between ROS production and depuration that favors a pro-oxidative environment, resulting from increased ROS production, decreased antioxidant defenses, or both. NIS expression and function are regulated by the cellular redox state in cancer and non-cancer contexts. In addition, OS has been implicated in thyroid tumorigenesis and thyroid cancer cell dedifferentiation. Here, we review the main aspects of redox homeostasis in thyrocytes and discuss potential ROS-dependent mechanisms involved in NIS repression in thyroid cancer.

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.

AMP-activated protein kinase (AMPK) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism and cell survival. Recently, our group described that AMPK plays an important role in the regulation of iodide and glucose uptake in thyroid cells. However, AMPK signaling pathway in human thyroid carcinomas has not been investigated so far. Objective To evaluate the expression and activity of AMPK in papillary thyroid carcinomas. Methods We examined total and phosphorylated AMPK (tAMPK and pAMPK) and phosphorylated acetyl-CoA-carboxylase (pACC) expressions through imunohistochemistry, using a tissue microarray block composed of 73 papillary thyroid carcinomas (PAP CA) or microcarcinomas (PAP MCA) and six adenoma (AD) samples from patients followed at the Federal University Hospital. The expression levels were compared with the non-neoplastic tissues from the same patient. Two different pathologists analyzed the samples and attributed scores of staining intensity and the proportion of stained cells. A total index was obtained by multiplying the values of intensity and the proportion of stained cells (INTxPROP). Results tAMPK, pAMPK, and pACC showed a predominant cytoplasmic staining in papillary carcinomas, adenomas, and non-neoplastic thyroid tissues. However, the intensity and the proportion of stained cells were higher in carcinomas, so that a significant increase was found in the INTxPROP score both in PAP CA and PAP MCA, when compared with their respective controls. Conclusion Our results show unequivocally that AMPK pathway is highly activated in papillary thyroid carcinomas; however, more studies are necessary to understand the pathophysiological significance of AMPK activation in thyroid carcinogenesis. Abstract AMP-activated protein kinase (AMPK) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism and cell survival. Recently, our group described that AMPK plays an important role in the regulation of iodide and glucose uptake in thyroid cells. However, AMPK signaling pathway in human thyroid carcinomas has not been investigated so far. Objective To evaluate the expression and activity of AMPK in papillary thyroid carcinomas. Methods We examined total and phosphorylated AMPK (tAMPK and pAMPK) and phosphorylated acetyl-CoA-carboxylase (pACC) expressions through imunohistochemistry, using a tissue microarray block composed of 73 papillary thyroid carcinomas (PAP CA) or microcarcinomas (PAP MCA) and six adenoma (AD) samples from patients followed at the Federal University Hospital. The expression levels were compared with the non-neoplastic tissues from the same patient. Two different pathologists analyzed the samples and attributed scores of staining intensity and the proportion of stained cells. A total index was obtained by multiplying the values of intensity and the proportion of stained cells (INTxPROP). Results tAMPK, pAMPK, and pACC showed a predominant cytoplasmic staining in papillary carcinomas, adenomas, and non-neoplastic thyroid tissues. However, the intensity and the proportion of stained cells were higher in carcinomas, so that a significant increase was found in the INTxPROP score both in PAP CA and PAP MCA, when compared with their respective controls. Conclusion Our results show unequivocally that AMPK pathway is highly activated in papillary thyroid carcinomas; however, more studies are necessary to understand the pathophysiological significance of AMPK activation in thyroid carcinogenesis.

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.