Final Report Summary - THYROIDANTIOXIDANT (Role of the Keap1/Nrf2 antioxidant response system in thyroid gland homeostasis and thyroid cancer) Background, Rationale and Objectives of the ProjectOxidative stress (OS) ensues when pro-oxidant and electrophilic reactive species overwhelm the cell’s antioxidant and detoxification proteins. In addition to causing protein and lipid damage, OS can cause mutations and epigenetic perturbation, and it is therefore a causative or exacerbating factor in a range of human disorders like cancer, neurodegeneration, ischemia-reperfusion injury, chronic obstructive pulmonary disease, and many others. Disorders linked to oxidation affect tissues that are exposed to the environment (skin, lung, digestive tract), generate high amounts of free radicals (muscle), function in detoxification (liver, kidney, placenta), or are particularly stress-sensitive (neurons). In contrast, little was yet known about the role of OS in the thyroid gland. This gap in knowledge was surprising, given that the thyroid generates on a daily basis high amounts of the oxidant hydrogen peroxide, which it uses to oxidize iodide and iodinate thyroglobulin in the process of thyroid hormone synthesis. It had been shown that, compared to other tissues, the thyroid has increased capacity for defending itself against OS (1). Specific antioxidant and detoxification enzymes had been identified that presumably help thyroid cells to maintain their homeostasis by ameliorating oxidative insults. While a minimal oxidative load is a prerequisite for normal thyroid cell function (2, 3), the gland’s antioxidant defense is activated by intra-thyroidal OS occurring during iodine deficiency, goitrogenesis, and high iodine-induced involution (4-6). However, the precise mechanisms by which thyroid cells sense and respond to OS remained unknown.The pathway centered on the transcription factor Nrf2 (NFE2-related transcription factor 2) was an excellent candidate for mediating the antioxidant response of the thyroid. In the absence of OS, Nrf2 binds to its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1), a protein tethered to the actin cytoskeleton (7). Keap1 suppresses the activity of Nrf2 both passively by sequestering it in the cytoplasm, as well as actively by targeting it for polyubiquitination and thus facilitating its proteasomal degradation. In addition to serving as an inhibitor of Nrf2, Keap1 also functions as a sensor of oxidants and electrophiles, which react with its redox-sensitive cysteine residues. Oxidative stressors or electrophilic xenobiotics abolish the inhibition of Nrf2 by Keap1. Nrf2 then accumulates in the nucleus where it transcriptionally activates protective genes through antioxidant response elements (AREs) in their regulatory sequences. Extensive research in organisms across the evolutionary spectrum has shown that Nrf2 defends animals against OS by preventing DNA and protein damage and protecting against stress-related pathologies (8). In animal models of disease, pharmacological activation of Nrf2 by various synthetic and natural compounds can prevent cancer and other disorders linked to OS. Since aging is also associated with oxidation, Nrf2 is required for normal lifespan and its induction can promote longevity. Both up-regulation and down-regulation of Nrf2 have been associated with human disease: Nrf2 activity is unexpectedly suppressed during normal aging and during the progression of OS-related disorders, due to changes in the abundance of Keap1 and other regulators. This paradoxical suppression of Nrf2 is thought to initiate a vicious cycle that further promotes organism and/or tissue aging and facilitates the clinical manifestation of aging- and stress-related diseases (8). Conversely, constitutive activation of Nrf2 by somatic mutations or epigenetic events that impair its interaction with Keap1 has been found in a variety of human cancers and promotes resistance to chemotherapy. Thus, tight regulation of Nrf2 activity is crucial for tissue homeostasis and disease prevention.Taken together, the global importance of Nrf2 for antioxidant defense; its high expression in the thyroid (http://biogps.gnf.org); and the association of iodine deficiency with OS, suggested that the Keap1/Nrf2 pathway could be critical for thyroid gland homeostasis, and that its inappropriate activation could be associated with thyroid cancer. Thus, the objectives of the project were to elucidate the role of the Keap1/Nrf2 antioxidant response pathway in the homeostasis of the thyroid gland under normal and goitrogenic or other pathological conditions, and to document its dysregulation by pathogenic mutations and/or epigenetic events in thyroid cancer.1. Maier J. et al. (2006) Endocrinology 147, 3391-3397; 2. Poncin, S. et al. (2009) J Endocrinol 201, 161-167; 3. Poncin, S. et al. (2010) Am J Pathol 176, 1-9; 4. Maier, J. et al. (2007) Biochim Biophys Acta 1773, 990-999; 5. Poncin, S. et al. (2008) Endocrinology 149, 424-433; 6. Krohn, K. et al. (2007) Nat Clin Pract Endocrinol Metab 3, 713-720 ; 7. Motohashi, H. et al. (2004) Trends Mol Med 10, 549-557; 8. Sykiotis, G. P. et al. (2010) Science Signaling 3, r3; 9. Eszlinger, M. et al. (2007) Curr Opin Endocrinol Diabetes Obes 14, 393-397.ResultsAim1 was to document Nrf2 signaling as regulator of thyroid antioxidant defense, and this goal has been achieved through work in two rodent cell lines (PCCL3 and FRTL5) and one human thyroid cell line (TAD2), which were used to investigate whether Nrf2 regulates antioxidant defenses in thyrocytes. Importantly, the cell lines were genotyped and found to be free of mutations in the genes encoding Nrf2 and Keap1. The Nrf2 pathway was manipulated experimentally with known inducers available commercially (sulforaphane, oltipraz) and with genetic constructs obtained or generated in-house to over-express or knock-down Nrf2 and Keap1. The results consistently showed that pathway activation increases resistance to oxidative insults, whereas its inhibition increases the sensitivity of the cells to oxidants and compromises their survival. In addition, Nrf2 was found to control the expression of genes fundamental for thyroid hormone synthesis, including TG, TSHR, TPO and NIS. These results have been presented in scientific conferences and a publication is in preparation.Aim 2 was to investigate the role of Nrf2 during conditions of altered thyroid physiology with the expectation that Nrf2 ameliorates intra-thyroidal OS. To this end, wild type and nrf2-/- mice were subjected to an established model of experimental goitrogenesis (treatment with the anti-thyroid medication PTU plus deprivation of iodine from the diet). Molecular analyses and immunohistochemistry (IHC) demonstrated that the Nrf2 pathway was activated by the treatment, thus verifying the hypothesis. However, there was no difference in the size of the goiters or the degree of hypothyroidism between wild-type and nrf2-/- animals, questioning whether Nrf2 is critical for the prevention of goiter in this model. Moreover, the administration of a goiter-rescuing chemical (prostaglandin J2) was not found to activate Nrf2. A potential explanation for these results is that goitrogenesis in this model is driven primarily by thyroid-stimulating hormone (TSH), which is secreted by the pituitary in high amounts in response to treatment with PTU (in this sense, this is a model of drug-induced primary hypothyroidism), and Nrf2 may not be related to TSH-driven processes. It was therefore postulated that Nrf2 might be functionally relevant for milder models of thyroid function perturbation, which do not involve TSH: the so called “thyroid autoregulation” refers to the adaptation of the gland to changes in the amount of available iodine, either shortage in the diet, or exposure to pharmacological doses, such as during a computed tomography (CT) scan with intravenous contrast (which invariably contains iodine). Indeed, further work showed that iodine exposure activates Nrf2 (molecular analyses of target genes and IHC for Nrf2), and Nrf2 was found to control the expression of genes fundamental for thyroid hormone synthesis, including TG, TSHR, TPO and NIS. Taken together, these data indicate that Nrf2 is critical for thyroid autoregulation. Lastly, Nrf2 was found to cross-talk with other stress protective cellular systems, such as the ubiquitin-proteasome pathway (UPS) and the unfolded protein response (UPR) during iodine exposure. These results have been presented in scientific conferences and a publication is in preparation.Aim 3 was to test whether constitutive Nrf2 activation is associated with human thyroid cancer. Although it was expected that thyroid cancers harbor somatic mutations in KEAP1 and NRF2, the analysis of archived thyroid cancer samples from patients did not reveal such mutations. Consistently, no such mutations were found in a panel of 10 human thyroid cancer cell lines. Nevertheless, immunohistochemical staining for Nrf2 and its target genes demonstrated that the pathway is indeed activated in thyroid cancer compared to control samples of healthy thyroid tissue from the same patients, as well as benign overgrowths (hyperplastic goiter) from unrelated patients. The control samples showed very low abundance of Nrf2, consistent with the hypothesis that pathway activation is a tumor-specific phenomenon. Therefore, pathway activation is not due to mutations in the pathway itself, as originally hypothesized, but due to other genetic or epigenetic events. In addition, it was shown that knock-down of Nrf2 in thyroid cancer cell lines is highly effective in reducing cell viability.Conclusions and Potential ImpactThe main conclusions from the project are that: (i) the Nrf2 antioxidant response pathway controls antioxidant genes in the thyroid and protects thyrocytes from cellular stress independently and via interaction with other proteostatic systems; (ii) Nrf2 regulates the expression of thyroid-specific genes with roles in thyroid hormone synthesis and thyroid autoregulation in response to changes in iodine availability; and (iii) increased activity of Nrf2 is a hallmark of papillary thyroid carcinoma because it protects transformed thyrocytes from oxidative stress.Correspondingly, the main implications of these findings are that: (i) understanding the antioxidant and iodine-handling properties of thyrocytes under various physiological and pathological conditions necessitates addressing the involvement of Nrf2; (ii) modeling and manipulating the Nrf2 system may help elucidate the molecular mechanisms of thyroid autoregulation, a fundamental physiological phenomenon with major clinical implications (iodine-induced hyper- and hypothyroidism) that is known for over 40 years but whose basis remains obscure; and (iii) Nrf2-inhibiting compounds, which are currently in development by several companies, might be useful in treating radioiodine-refractory or metastatic thyroid cancer. Thus, the results of the project are of interest to basic researchers, translational investigators (including endocrinologists), patients affected by thyroid disorders, and SMEs and pharmaceutical companies pursuing the Nrf2 pathway as a drug target.Project website and contact informationThe project’s website is live and will be maintained indefinitely: www.thyroid.eu. A “.eu” address was specifically chosen to highlight the European dimension and character of the project. The support from the Marie S. Curie scheme is explicitly acknowledged on the website. Links to the published scientific results of the project are featured on the website, which contains general information about the project as well as contact information. The website will be updated with the expected future publications.