A produção mundial de carne bovina corresponde a 58,856 milhões de toneladas, das quais 9,92 milhões de toneladas de carne são produzidas pelo Brasil. Com o maior rebanho bovino comercial (aproximadamente 209 milhões de bovinos), nosso país ocupa posição de destaque no mercado da carne bovina: 1º em exportação (2,03 milhões de toneladas) e 2º em produção e consumo (40 kg/habitante/ano). Ao longo dos anos a produção de carne bovina aumentou, assim como as críticas no campo da saúde humana devido ao seu alto teor de ácidos graxos saturados e a possível, mas ainda questionável relação com doenças cardiovasculares, diabetes e câncer. Por estas razões uma série de pesquisas tem sido realizada na tentativa de alterar a composição do perfil de ácidos graxos da carne, o que consequentemente afeta sua estabilidade oxidativa . Para avaliar a influência de componentes específicos da dieta sobre as respostas do organismo e seus efeitos sobre a produção e a qualidade de carne, tem se avaliado a expressão individual de genes, ou seja, tem se utilizado a transcriptômica. A partir do uso desta ferramenta, o presente estudo avaliou a interação entre a suplementação com selênio, vitamina E e/ou óleo de canola e a expressão de genes-chave na regulação das vias antioxidante, lipogênica e colesterolêmica na espécie bovina. Bovinos Nelore (n=48) não castrados, peso inicial ±350 kg, alocados em calan gate nas dependências do Departamento de Zootecnia da FZEA-USP Pirassununga, SP foram divididos em 4 grupos : Controle (C): dieta base; (Se + Vit E): 2,5 mg de Se e 500 UI de vitamina E /kg de MS; (Canola): adição de 3% de óleo de canola/kg de MS; (Se + Vit E + Canola): adição de 2,5mg de Se, 500 UI de vitamina E e 3% de óleo de canola /kg de MS. Ao final de 12 semanas, foram abatidos (peso final ±456 kg) e amostras de fígado, músculo L. dorsi e tecido adiposo subcutâneo coletadas para análise da expressão gênica de NFE2L2, GPX1, GPX4, GSR, GCLG, GSS, SREBF1, PPARA, SCD, HMGCR, ACAT1, ACAT2 e LCAT. Os dados foram analisados pelo PROC MIXED SAS (2005), as médias foram comparadas por meio de contrastes avaliados pelo teste t de Student e a correlação entre as expressões gênicas hepáticas foram estimadas por meio do coeficiente de correlação de Pearson. O tecido hepático foi mais sensível às mudanças na taxa de transcrição de mRNA. Os animais que receberam a suplementação com Se junto a Vitamina E apresentaram maior expressão de genes citoprotetores (NFE2L2, P≤0,05; GPX1, P≤0,08; GPX4, P≤0,03; GSS, P≤0,06), lipogênico (SCD, P≤0,06) e colesterolêmico (HMGCR, P≤0,06) em relação aos que receberam o óleo de canola. Estes, por sua vez, apresentaram maior quantidade de mRNA para LCAT (P≤0,05) em relação aos que receberam suplementação com antioxidantes. As expressões gênicas de SREBF1 (lipogênico; P≤0,03) e ACAT1 (colesterolêmico; P≤0,08) foram maiores para os animais que receberam estes antioxidantes associados ao óleo de canola em comparação aos que receberam tais nutrientes de forma segregada. Os resultados estimulam estudos com estes antioxidantes e fontes de ácidos graxos poli-insaturados como o óleo de canola diante do potencial destes, seja para a qualidade da carne, seja para uso terapêutico na saúde animal ou do homem. Contudo, é necessário cautela, pois estes antioxidantes podem tanto induzir genes citoprotetores, quanto genes lipogênicos e colesterolêmicos e, a maneira pela qual tal ativação ocorre sugere a participação do NFE2L2. / World beef production corresponds to 58.856 million tons, of which 9.92 million tons of meat are produced by Brazil. With the largest commercial cattle herd (approximately 209 million animals), our country occupies a prominent position in the beef market: 1st in exports (2.03 million tons) and 2nd in production and consumption (40 kg / capita /year). Over the years, beef production increased, as critical in the field of human health due to its high content of saturated fatty acids and a possible but still questionable relationship with cardiovascular disease, diabetes and cancer. For these reasons a lot of research has been conducted in an attempt to change the composition of the meat fatty acids, which consequently affects the oxidative stability of the meat. To evaluate the influence of specific dietary components on the responses of the body and its effects on the production and quality of meat, has evaluated the expression of individual genes, or has used transcriptomics. From the use of this tool, the present study evaluated the interaction between supplementation with selenium, vitamin E and/or canola oil and the expression of key genes in the regulation of antioxidant, lipogenic and cholesterolemic pathways in cattle. Nelore (n= 48) uncastrated, initial weight ± 350kg, allocated in calan gate in the Department of Animal Science, FZEA Pirassununga-USP, SP were divided into 4 groups: Control (C): basic diet; (Se + Vit E): 2.5 mg Se and 500 IU vitamin E/kg DM; (Canola): addition of 3% canola/kg of DM oil; (Se + Vit E + Canola): adding 2.5 mg Se, 500 IU vitamin E and 3% canola/kg of DM oil. After 12 weeks, they were slaughtered (final weight ± 456 kg) and samples of liver, L. dorsi muscle and subcutaneous adipose tissue collected for analysis of gene expression of NFE2L2, GPX1, GPX4, GSR, GCLG, GSS, SREBF1, PPARA, SCD, HMGCR, ACAT1, ACAT2 and LCAT. Data were analyzed using PROC MIXED (SAS 2005), the means were compared by contrasts evaluated by Student\'s t test and the correlation between hepatic gene expression were estimated using the Pearson correlation coefficient. The liver tissue was more sensitive to changes in the rate of transcription of mRNA. Animals that received supplementation with Vitamin E and Se had higher expression of cytoprotective genes (NFE2L2, P≤0.05; GPX1, P≤0.08; GPX4, P≤0.03; GSS, P≤0.06), lipogenic (SCD, P≤0.06) and cholesterolemic (HMGCR, P≤0.06) compared to those receiving canola oil. These, in turn, showed a higher amount of mRNA for LCAT (P≤0.05) than those who received supplementation with antioxidants. The gene expressions of SREBF1 (lipogenic, P≤0.03) and ACAT1 (cholesterolemic, P≤0.08) were higher for animals receiving these antioxidants associated with canola oil compared to those which received these nutrients isolated. These results stimulate more studies about antioxidants and polyunsaturated fatty acids such as canola oil because of their potential either for the quality of the meat, either for therapeutic use in animal or human health. However, caution is needed because these antioxidants can both induce cytoprotective genes, as well as lipogenic and cholesterolemic genes, and the way this activation occurs suggests the involvement of NFE2L2.

A produção mundial de carne bovina corresponde a 58,856 milhões de toneladas, das quais 9,92 milhões de toneladas de carne são produzidas pelo Brasil. Com o maior rebanho bovino comercial (aproximadamente 209 milhões de bovinos), nosso país ocupa posição de destaque no mercado da carne bovina: 1º em exportação (2,03 milhões de toneladas) e 2º em produção e consumo (40 kg/habitante/ano). Ao longo dos anos a produção de carne bovina aumentou, assim como as críticas no campo da saúde humana devido ao seu alto teor de ácidos graxos saturados e a possível, mas ainda questionável relação com doenças cardiovasculares, diabetes e câncer. Por estas razões uma série de pesquisas tem sido realizada na tentativa de alterar a composição do perfil de ácidos graxos da carne, o que consequentemente afeta sua estabilidade oxidativa . Para avaliar a influência de componentes específicos da dieta sobre as respostas do organismo e seus efeitos sobre a produção e a qualidade de carne, tem se avaliado a expressão individual de genes, ou seja, tem se utilizado a transcriptômica. A partir do uso desta ferramenta, o presente estudo avaliou a interação entre a suplementação com selênio, vitamina E e/ou óleo de canola e a expressão de genes-chave na regulação das vias antioxidante, lipogênica e colesterolêmica na espécie bovina. Bovinos Nelore (n=48) não castrados, peso inicial ±350 kg, alocados em calan gate nas dependências do Departamento de Zootecnia da FZEA-USP Pirassununga, SP foram divididos em 4 grupos : Controle (C): dieta base; (Se + Vit E): 2,5 mg de Se e 500 UI de vitamina E /kg de MS; (Canola): adição de 3% de óleo de canola/kg de MS; (Se + Vit E + Canola): adição de 2,5mg de Se, 500 UI de vitamina E e 3% de óleo de canola /kg de MS. Ao final de 12 semanas, foram abatidos (peso final ±456 kg) e amostras de fígado, músculo L. dorsi e tecido adiposo subcutâneo coletadas para análise da expressão gênica de NFE2L2, GPX1, GPX4, GSR, GCLG, GSS, SREBF1, PPARA, SCD, HMGCR, ACAT1, ACAT2 e LCAT. Os dados foram analisados pelo PROC MIXED SAS (2005), as médias foram comparadas por meio de contrastes avaliados pelo teste t de Student e a correlação entre as expressões gênicas hepáticas foram estimadas por meio do coeficiente de correlação de Pearson. O tecido hepático foi mais sensível às mudanças na taxa de transcrição de mRNA. Os animais que receberam a suplementação com Se junto a Vitamina E apresentaram maior expressão de genes citoprotetores (NFE2L2, P≤0,05; GPX1, P≤0,08; GPX4, P≤0,03; GSS, P≤0,06), lipogênico (SCD, P≤0,06) e colesterolêmico (HMGCR, P≤0,06) em relação aos que receberam o óleo de canola. Estes, por sua vez, apresentaram maior quantidade de mRNA para LCAT (P≤0,05) em relação aos que receberam suplementação com antioxidantes. As expressões gênicas de SREBF1 (lipogênico; P≤0,03) e ACAT1 (colesterolêmico; P≤0,08) foram maiores para os animais que receberam estes antioxidantes associados ao óleo de canola em comparação aos que receberam tais nutrientes de forma segregada. Os resultados estimulam estudos com estes antioxidantes e fontes de ácidos graxos poli-insaturados como o óleo de canola diante do potencial destes, seja para a qualidade da carne, seja para uso terapêutico na saúde animal ou do homem. Contudo, é necessário cautela, pois estes antioxidantes podem tanto induzir genes citoprotetores, quanto genes lipogênicos e colesterolêmicos e, a maneira pela qual tal ativação ocorre sugere a participação do NFE2L2. / World beef production corresponds to 58.856 million tons, of which 9.92 million tons of meat are produced by Brazil. With the largest commercial cattle herd (approximately 209 million animals), our country occupies a prominent position in the beef market: 1st in exports (2.03 million tons) and 2nd in production and consumption (40 kg / capita /year). Over the years, beef production increased, as critical in the field of human health due to its high content of saturated fatty acids and a possible but still questionable relationship with cardiovascular disease, diabetes and cancer. For these reasons a lot of research has been conducted in an attempt to change the composition of the meat fatty acids, which consequently affects the oxidative stability of the meat. To evaluate the influence of specific dietary components on the responses of the body and its effects on the production and quality of meat, has evaluated the expression of individual genes, or has used transcriptomics. From the use of this tool, the present study evaluated the interaction between supplementation with selenium, vitamin E and/or canola oil and the expression of key genes in the regulation of antioxidant, lipogenic and cholesterolemic pathways in cattle. Nelore (n= 48) uncastrated, initial weight ± 350kg, allocated in calan gate in the Department of Animal Science, FZEA Pirassununga-USP, SP were divided into 4 groups: Control (C): basic diet; (Se + Vit E): 2.5 mg Se and 500 IU vitamin E/kg DM; (Canola): addition of 3% canola/kg of DM oil; (Se + Vit E + Canola): adding 2.5 mg Se, 500 IU vitamin E and 3% canola/kg of DM oil. After 12 weeks, they were slaughtered (final weight ± 456 kg) and samples of liver, L. dorsi muscle and subcutaneous adipose tissue collected for analysis of gene expression of NFE2L2, GPX1, GPX4, GSR, GCLG, GSS, SREBF1, PPARA, SCD, HMGCR, ACAT1, ACAT2 and LCAT. Data were analyzed using PROC MIXED (SAS 2005), the means were compared by contrasts evaluated by Student\'s t test and the correlation between hepatic gene expression were estimated using the Pearson correlation coefficient. The liver tissue was more sensitive to changes in the rate of transcription of mRNA. Animals that received supplementation with Vitamin E and Se had higher expression of cytoprotective genes (NFE2L2, P≤0.05; GPX1, P≤0.08; GPX4, P≤0.03; GSS, P≤0.06), lipogenic (SCD, P≤0.06) and cholesterolemic (HMGCR, P≤0.06) compared to those receiving canola oil. These, in turn, showed a higher amount of mRNA for LCAT (P≤0.05) than those who received supplementation with antioxidants. The gene expressions of SREBF1 (lipogenic, P≤0.03) and ACAT1 (cholesterolemic, P≤0.08) were higher for animals receiving these antioxidants associated with canola oil compared to those which received these nutrients isolated. These results stimulate more studies about antioxidants and polyunsaturated fatty acids such as canola oil because of their potential either for the quality of the meat, either for therapeutic use in animal or human health. However, caution is needed because these antioxidants can both induce cytoprotective genes, as well as lipogenic and cholesterolemic genes, and the way this activation occurs suggests the involvement of NFE2L2.

Θεωρητικό υπόβαθρο: Το οξειδωτικό στρες (ΟΣ) ορίζεται ως το παθολογικό αποτέλεσμα που προκύπτει από τη διαταραχή της ισορροπίας των κυτταρικών συγκεντρώσεων των οξειδωτικών, δραστικών ενώσεων και των αντιοξειδωτικών μορίων. Εκτός από τη βλάβη που υπόκεινται οι πρωτεΐνες και τα λιπίδια, το ΟΣ μπορεί επίσης να προκαλέσει μεταλλάξεις και επιγενετικές μεταβολές καταστρέφοντας τόσο το DNA όσο και τις πρωτεΐνες που τροποποιούν τη χρωματίνη. Παρ' όλα αυτά, στα θυρεοειδικά θυλακικά κύτταρα παράγονται σε καθημερινή βάση υψηλές ποσότητες υπεροξειδίου του υδρογόνου (H2O2), οξειδωτικής ουσίας απαραίτητης για την πραγματοποίηση της θυρεοειδικής ορμονογένεσης. Δεδομένου ότι ένα ελάχιστο ποσό οξειδωτικού φορτίου αποτελεί προϋπόθεση αφ’ενός για τη φυσιολογική λειτουργία των θυλακικών κυττάρων και αφ’ετέρου για την ανάπτυξη του θυρεοειδούς αδένα, πρόσφατα αποδείχτηκε ότι ο θυρεοειδής αδένας παρουσιάζει αυξημένη αμυντική ανταπόκριση έναντι του ΟΣ. Ωστόσο, οι ακριβείς μηχανισμοί με τους οποίους τα θυλακικά κύτταρα αντιλαμβάνονται και απαντούν στο ΟΣ παραμένουν ασαφείς. Ο NFE2-related factor 2 (Nrf2), ο οποίος κωδικοποιείται από το γονίδιο NFE2L2, είναι ένας μεταγραφικός παράγοντας ο οποίος απαντά σε σήματα κυτταρικού στρες και ανταποκρίνεται επιδρώντας στη μεταγραφή γονιδίων σε διάφορους τύπους ιστών. Σε βασικές συνθήκες, ο Nrf2 οδηγείται σε πρωτεασωματική αποικοδόμηση μέσω του κυτταροπλασματικού του αναστολέα, Keap1, ενώ σε συνθήκες ΟΣ, η αποικοδόμηση του Nrf2 δεν είναι δυνατή και ο Nrf2 εισέρχεται στον πυρήνα ώστε να ενεργοποιήσει τη μεταγραφή αντιοξειδωτικών γονιδίων όπως του γονιδίου Nqo1. Καθώς η οξειδοαναγωγική ομοιοστασία κατέχει κεντρικό ρόλο στην φυσιολογία του θυρεοειδούς αδένα και ο Nrf2 πρόσφατα χαρακτηρίσθηκε ως μεσολαβητής στην αντίσταση θυρεοειδικών καρκινικών κυτταρικών σειρών σε πρωτεασωμικούς αναστολείς, το αντιοξειδωτικό μονοπάτι Nrf2 μπορεί να θεωρηθεί ως εξαιρετικός υποψήφιος της διαμεσολάβησης της απόκρισης του θυρεοειδούς αδένα στο ΟΣ. Παρ 'όλα αυτά, ο ρόλος του μονοπατιού Nrf2 στον ανθρώπινο θυρεοειδικό καρκίνο παραμένει άγνωστος. Στόχος: Στόχοι της παρούσας μελέτης ήταν η εκτίμηση της δραστηριότητας του μονοπατιού Νrf2 στο διαφοροποιημένο καρκίνωμα του θυρεοειδούς αδένα και η διερεύνηση σωματικών μεταλλάξεων των γονιδίων NFE2L2 και Keap1. Yλικά και Μέθοδοι Ασθενείς: Στη μελέτη συμμετείχαν 90 περιστατικά εκ των οποίων τα 42 αφορούσαν θηλώδη καρκινώματα (papillary thyroid carcinomas, PTCs), τα 6 θυλακιώδη καρκινώματα (follicular thyroid carcinomas, FTCs) και τα υπόλοιπα 42 καλοήθεις όγκους (24 αδενώματα και 18 οζώδης υπερπλασία). Κυτταρικές σειρές: Στα πλαίσια της παρούσας μελέτης χρησιμοποιήθηκαν κυτταρικές σειρές PTC (K1, TPC-1, XTC-1), κυτταρική σειρά φτωχά διαφοροποιημένου PTC (T243), κυτταρικές σειρές αδιαφοροποίητου καρκινώματος (C643, 8505C, Hth74) και τέλος κυτταρικές σειρές αναπλαστικού καρκινώματος (T235 , T241, T238). Μέθοδοι: Αναδρομική ανοσοϊστοχημική ανάλυση δειγμάτων PTC και FTC, παρακείμενου φυσιολογικού ιστού και καλοηθών βλαβών. Ανάλυση αλληλουχίας DNA των κυτταρικών σειρών και PTC δειγμάτων. Κύριες μετρήσεις και υπολογισμοί: Αξιολογήθηκε η ένταση της ανοσοαντίδρασης των δειγμάτων των ιστών σε αντισώματα για τα Nrf2, Nqo1, Keap1 και 4-HNE. Μελετήθηκε η αλληλουχία του εξονίου 2 του γονιδίου NFE2L2 καθώς και του γονιδίου Keap1. Αποτελέσματα: O μεταγραφικός παράγοντας Nrf2 καθώς και ο στόχος του, η πρωτεΐνη Nqo1 ήταν μη ανιχνεύσιμα σε φυσιολογικό ιστό θυρεοειδούς αδένα. Τα επίπεδά τους ήταν σημαντικά υψηλότερα στα PTC δείγματα από ό,τι στα δείγματα καλοηθών βλαβών. Η έκφραση του Keap1 εμφάνισε διακύμανση στα δείγματα PTC με τα επίπεδά του να μην εμφανίζουν συσχέτιση με τα αντίστοιχα του Nrf2, ενάντια στη θεωρία πως τα μειωμένα επίπεδα του Κeap1 συνιστούν μηχανισμό ενεργοποίησης του Nrf2. Ο δείκτης ΟΣ, 4-HNE βρέθηκε αυξημένος στη πλειοψηφία των δειγμάτων PTC σε σχέση με το φυσιολογικό ιστό αναδεικνύοντας την ύπαρξη αυξημένου ΟΣ στο PTC. Επιπλέον, όσον αφορά τα δείγματα FTC, ο μεταγραφικός παράγοντας Nrf2 και η πρωτεΐνη Nqo1 ήταν ανιχνεύσιμα σε όλα τα δείγματα, ενώ τα επίπεδα του 4-ΗΝΕ ήταν αυξημένα. Όσον αφορά την ανάλυση αλληλουχίας DNA στις καρκινικές σειρές και σε 11 δείγματα PTC με υψηλή έκφραση Nrf2, καμία μετάλλαξη δεν ανευρέθηκε στο εξόνιο 2 του γονιδίου NFE2L2 και στο γονίδιο Keap1. Συμπεράσματα: Τα αποτελέσματα της μελέτης μας σε συνδυασμό με περαιτέρω μελέτες από το εργαστήριο Ενδοκρινολογίας και Ανατομικής του Πανεπιστημίου Πατρών καθώς και από το BC κέντρο έρευνας καρκίνου (Vancouver, Canada) αποδεικνύουν ότι το μονοπάτι Nrf2 ενεργοποιείται σε PTC και κατέχει ρυθμιστικό ρόλο στην αντιοξειδωτική απόκριση και τη βιωσιμότητα των θυρεοειδικών καρκινικών κυττάρων. Συνεπώς, αναδεικνύεται το Nrf2 μονοπάτι ως νέο “σήμα κατατεθέν” του PTC. Παρά το γεγονός ότι δεν ήταν δυνατή η πραγματοποίηση στατιστικών συσχετίσεων στη μελέτη του FTC λόγω του περιορισμένου αριθμού δειγμάτων, το μονοπάτι Nrf2 φαίνεται να ενεργοποιείται επίσης στο FTC. Η σταθερή ενεργοποίηση του Nrf2 στο PTC και ενδεχομένως στο FTC δίνει το έναυσμα για περαιτέρω διερεύνηση του μονοπατιού αυτού σε όλα τα είδη θυρεοειδικού καρκίνου καθώς και της πιθανής διαγνωστικής, προγνωστικής, και/ή θεραπευτικής χρησιμότητας του μονοπατιού στο διαφοροποιημένο καρκίνο του θυρεοειδούς αδένα. / Scientific background: Oxidative stress (ΟS) is experienced by cells when pro-oxidant and electrophilic reactive species overwhelm the cell’s antioxidant and detoxification proteins. In addition to causing protein and lipid damage, oxidative stress can cause mutations and epigenetic perturbation by damaging DNA and proteins that modify chromatin. Nevertheless, in thyrocytes a daily basis high amounts of the oxidant hydrogen hyperoxide (H2O2) was generated due to the fact that H2O2 is a reactive oxygen species required for thyroid hormonogenesis. A minimal oxidative load is a prerequisite for normal thyroid cell function and development and it was recently shown that the thyroid has increased capacity for defending itself against OS. However, precise mechanisms by which thyrocytes sense and respond to OS remain obscure. NFE2-related factor 2 (Nrf2), encoded by NFE2L2 gene, is a transcription factor that integrates cellular stress signals and responds by directing transcriptional program in various tissues. In basal conditions, Nrf2 is targeted for proteasomal degradation by its cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), while in oxidative stress Nrf2 degradation is abolished and Nrf2 accumulates in the nucleus where it transactivates protective genes such as NAD(P)H dehydrogonase quinone 1 (Nqo1). As redox homeostasis plays a principal role in thyroid gland’s physiology, and Nrf2 has recently been characterised as mediator of thyroid cancer cell lines’ resistance to proteasome inhibitors, the Nrf2 antioxidant pathway seems to be an excellent candidate for mediating the antioxidant response of the thyroid gland. Nevertheless, the activity status of the Nrf2 pathway in human thyroid cancer remains unknown. Objective: The aims of this study were to assess the activity status of the Nrf2 pathway in differentiated thyroid carcinoma and investigate somatic mutations in NFE2L2 and Keap1 genes. Μethods and Materials Patients: The study included 90 individual samples; 42 papillarz thyroid carcinomas (PTCs), 6 follicular thyroid carcinomas (FTCs) and 42 benign lesions (24 adenomas and 18 nodular hyperplasias). Cell lines: Ten thyroid cell lines are used for this study: The PTC cell lines, K1, TPC-1 and XTC-1; the poorly differentiated PTC cell line, T243; the undifferentiated carcinoma cell lines, C643, 8505C and Hth74; and the anaplastic carcinoma cell lines, T235, T241 and T238. Methods: We conducted retrospective immunohistochemical analyses of PTC and FTC specimens, adjacent normal tissue, and benign lesions; DNA sequencing in cell lines and PTC samples. Main Outcome Measures: We assessed the abundance of Nrf2, Nqo1, Keap1, and 4HNE; and the sequence of NFE2L2 gene’s exon 2 and of KEAP1 gene. Results: Nrf2 and its target Nqo1 were undetectable in normal tissue; their levels were significantly higher in PTC than in benign lesions. The Nrf2 inhibitor, Keap1 was variably abundant in PTC, and its levels did not correlate with Nrf2, arguing against decreased levels as the mechanism for Nrf2 activation. The oxidized lipid 4HNE was more abundant in PTC than normal tissue indicating oxidative stress. In addition, as far as FTC samples are concerned, Nrf2 and Nqo1 were detectable in all samples as well as the levels of 4-HNE were significantly high. No mutations were detectable in exon 2 of NFE2L2 gene and in Keap1 gene. Conclusions: Our study’s results supported by further studies in laboratories of Endocrinology and Anatomy at University of Patras and BC Cancer Research Center (Vancouver, Canada) demonstrate that the Nrf2 pathway is commonly activated in PTC and that it regulates antioxidant responses and viability of cancer cells. Thus, Nrf2 is highlighted as a new hallmark of PTC. Although, statistic correlations were not possible in FTC samples’ study because of small sample size, the Nrf2 pathway seems to be also activated in FTC. The high activity of Nrf2 in PTC and possibly in FTC warrants further exploration of this pathway’s potential diagnostic, prognostic, and/or therapeutic utility in differentiated thyroid carcinoma.

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1(+/+) than in KEAP1(-/-) isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1(-/-) cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.