expressão do gene nr4a1 em carcinomas da tiróide e seu papel
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CLÉBER PINTO CAMACHO EXPRESSÃO DO GENE NR4A1 EM CARCINOMAS DA TIRÓIDE E SEU PAPEL POTENCIAL NA TERAPIA DO CÂNCER Dissertação apresentada à Universidade Federal de São Paulo para obtenção do título de Mestre em Ciências SÃO PAULO 2008 CLÉBER PINTO CAMACHO EXPRESSÃO DO GENE NR4A1 EM CARCINOMAS DA TIRÓIDE E SEU PAPEL POTENCIAL NA TERAPIA DO CÂNCER Dissertação apresentada à Universidade Federal de São Paulo para obtenção do título de Mestre em Ciências ORIENTADOR: Prof. Dr. Rui Monteiro de Barros Maciel CO-ORIENTADORA: Profa. Dra. Janete Maria Cerutti SÃO PAULO 2008 Camacho, Cléber Pinto EXPRESSÃO DO GENE NR4A1 EM CARCINOMAS DA TIRÓIDE E SEU PAPEL POTENCIAL NA TERAPIA DO CÂNCER, São Paulo, 2008 p. 41 Tese de Mestrado – Disciplina de Endocrinologia - Universidade Federal de São Paulo, Escola Paulista de Medicina Orientador: Prof. Dr. Rui Monteiro de Barros Maciel Co-orientadora: Profa. Dra. Janete Maria Cerutti Descritores: NR4A1, CCND1, FOSB, Câncer de Tiróide – Neoplasias Foliculares – Linhagens Celulares da Tiróide – Expressão Gênica – SAGE – PCR em Tempo Real – Lítio UNIVERSIDADE FEDERAL DE SÃO PAULO ESCOLA PAULISTA DE MEDICINA CURSO DE PÓS-GRADUAÇÃO EM ENDOCRINOLOGIA TESE DE MESTRADO AUTOR: Cléber Pinto Camacho ORIENTADOR: Prof. Dr. Rui Monteiro de Barros Maciel CO-ORIENTADOR: Profa Dra. Janete Maria Cerutti TÍTULO: EXPRESSÃO DO GENE NR4A1 EM CARCINOMAS DA TIRÓIDE E SEU PAPEL POTENCIAL NA TERAPIA DO CÂNCER BANCA EXAMINADORA: MEMBROS EFETIVOS: 1. Profa. Dra. Laura S. Ward 2. Profa. Dra. Léa Maria Zanini Maciel 3. Prof. Dr. Magnus Regios Dias da Silva MEMBRO SUPLENTE 1. Profa. Dra. Denise Pires de Carvalho Data: 28 de Novembro de 2007 À minha esposa, Dáfne, a quem devo os momentos alegres e o apoio nos momentos difíceis. Companheira que trilha comigo os mesmos caminhos, os mesmos desafios, dividindo problemas e sucessos. Aos meus pais, Manoel e Célia, a quem devo o esforço pela minha educação e o incentivo para seguir adiante, não importando a distância ou as pedras no caminho. À minha irmã, Keite, pela amizade e pela percepção diferenciada do mundo. Ao Prof. Dr. Rui M. B. Maciel, meu obrigado pelos desafios que me fizeram pensar. Pelas discussões que me fizeram aprender. Pelos ensinamentos que me fizeram saber ensinar. Muito obrigado pela amizade que me permitiu continuar. À Profa. Dra. Janete Maria Cerutti, um ícone de seriedade, dedicação e profissionalismo, a quem devo muito do meu aprendizado em biologia molecular e a ajuda nos passos dados durante o desenvolvimento desta tese. Agradecimentos Não seria possível listar todos aqueles que influenciaram minha vida para que ela convergisse neste caminho e neste trabalho, mas posso registrar aqui o agradecimento àqueles que mais diretamente participaram deste momento. Pessoas muito especiais que influenciaram a minha vida, das quais desejo para sempre me lembrar. Inicialmente gostaria de agradecer à Profª Drª Denise Câmara Lamego Martins, que me incentivou a seguir a vida acadêmica e despertou meu interesse inicial pela Tiroidologia. Agradeço a amizade dos colegas endocrinologistas Magnus, Rosa Paula, Claudia Nakabashi, Daniele e Maria Izabel, com quem convivi nos ambulatórios e nas bancadas, amigos que transformaram a assistência aos pacientes e a pesquisa em um aprendizado ainda mais rico. Não poderia me esquecer das endocrinologistas Maria da Conceição e Elza, que através do apoio e amizade me ajudaram a prosseguir. Agradeço também aos amigos do Laboratório de Endocrinologia Molecular: Adriana, Gustavo, Gisele, Flávia, Rosana, Mariana e Felipe; com os quais aprendi, através de acertos e erros, técnicas e protocolos. Amigos de bancada, dedicados à pesquisa, com os quais passei grande parte dos meus dias e, muitas vezes, noites, trabalhando na bancada, e com os quais comecei a enxergar um mundo invisível que nos cerca. Uma lembrança especial para Gisele Oler e Flávia Latini, pela colaboração e sugestões que foram muito importantes para o trabalho, amigas sem as quais este caminho teria sido mais tortuoso e muito menos divertido. Agradeço também aos amigos Teresa, Ilda e Gilberto, pelo ombro amigo sempre presente, pela compreensão nos momentos difíceis e pelas discussões que me ajudaram a resolver problemas durante a realização deste trabalho. Ao Prof. Dr. Reinaldo Furlanetto e à Profa Dra Luiza Matsumura, pelos ensinamentos, conselhos e discussões sobre a Endocrinologia e sobre a vida. Às secretárias Ângela, Yeda e Amarillys, pela atenção especial e carinho, pelos quais serão sempre lembradas. Aos alunos e residentes que me permitiram ensinar, e com os quais muito aprendí. A todos os pacientes, pela colaboração, pois sem eles este trabalho e os seus ensinamentos não teriam sentido. Ao Prof. Dr. Sérgio Atala Dib, coordenador da Pós-Graduação, pela atenção, discussões e todos os ensinamentos. À CAPES, pela contribuição financeira através da concessão da bolsa de mestrado. Índice APRESENTAÇÃO ___________________________________________________________i INTRODUÇÃO ____________________________________________________________1 OBJETIVOS ____________________________________________________________4 MANUSCRITO ____________________________________________________________5 CONCLUSÕES ____________________________________________________________27 BIBLIOGRAFIA ____________________________________________________________28 APRESENTAÇÃO Nesta tese de mestrado, realizada com apoio financeiro da CAPES e da FAPESP (05/60330-8), intitulada “EXPRESSÃO DO GENE NR4A1 EM CARCINOMAS DA TIRÓIDE E SEU PAPEL POTENCIAL NA TERAPIA DO CÂNCER” apresentamos, de acordo com as recomendações da comissão especial do programa de Pós-Graduação em Endocrinologia da Universidade Federal de São Paulo (UNIFESP), um trabalho em forma de manuscrito que será submetido à revista Clinical Endocrinology. O trabalho foi desenvolvido no laboratório de Endocrinologia Molecular da UNIFESP. Por orientação do Prof. Dr. Rui Maciel e da Profª Dra Janete Cerutti, estabelecemos como foco os tumores foliculares da tiróide, em particular o perfil de expressão gênica de suas células tumorais. A partir das bibliotecas geradas através da técnica SAGE, trabalho este da Profª Drª Janete Maria Cerutti, em colaboração com o Prof. Dr. Gregory J. Riggins, pudemos selecionar genes candidatos ao estudo (1). Todas as bibliotecas de Tags geradas estão disponíveis no site do Cancer Genome Anatomy Project (CGAP) para download ou análise - http://cgap.nci.nih.gov/SAGE. Identificamos genes cuja expressão estava diminuída nas bibliotecas de carcinoma folicular de tiróide (FTC): TPO, TFF3, NR4A1 e IYD. O NR4A1 foi selecionado para uma análise mais profunda, por apresentar expressão potencialmente tecido-específica. Passamos então à validação do padrão diferencial de expressão em tecidos neoplásicos e não-neoplásicos, pela técnica de PCR em tempo real, assim como a realização de experimentos em linhagens celulares, com o uso de lítio. Estes são os focos desta tese e estão descritos no manuscrito “Downregulation of NR4A1 in follicular thyroid carcinoma cell line is restored after lithium treatment”. i Durante todo o período em que estive desenvolvendo esta pesquisa, acompanhei e participei de vários outros trabalhos de teses relacionados com doenças malignas e benignas da tiróide e que resultaram em publicações onde sou co-autor (2-5). Reis EM, Ojopi EP, Alberto FL, Rahal P, Tsukumo F, Mancini UM, Guimarães GS, Thompson GM, Camacho C, Miracca E, Carvalho AL, Machado AA, Paquola AC, Cerutti JM, da Silva AM, Pereira GG, Valentini SR, Nagai MA, Kowalski LP, Verjovski-Almeida S, Tajara EH, Dias-Neto E, Bengtson MH, Canevari RA, Carazzolle MF, Colin C, Costa FF, Costa MC, Estécio MR, Esteves LI, Federico MH, Guimarães PE, Hackel C, Kimura ET, Leoni SG, Maciel RM, Maistro S, Mangone FR, Massirer KB, Matsuo SE, Nobrega FG, Nóbrega MP, Nunes DN, Nunes F, Pandolfi JR, Pardini MI, Pasini FS, Peres T, Rainho CA, dos Reis PP, Rodrigus-Lisoni FC, Rogatto SR, dos Santos A, dos Santos PC, Sogayar MC, Zanelli CF; Head and Neck Annotation Consortium. 2005 Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer. Cancer Res 65:1693-9 Biscolla RP, Ikejiri ES, Mamone MC, Nakabashi CC, Andrade VP, Kasamatsu TS, Crispim F, Chiamolera MI, Andreoni DM, Camacho CP, Hojaij FC, Vieira JG, Furlanetto RP, Maciel RM. 2007 [Diagnosis of metastases in patients with papillary thyroid cancer by the measurement of thyroglobulin in fine needle aspirate]. Arq Bras Endocrinol Metabol 51:419-25 Guimaraes GS, Latini FR, Camacho CP, Maciel RM, Dias-Neto E, Cerutti JM 2006 Identification of candidates for tumor-specific alternative splicing in the thyroid. Genes Chromosomes Cancer 45:540-53 ii Tamanaha R, Camacho CP, Ikejiri ES, Maciel RM, Cerutti JM 2007 Y791F RET mutation and early onset of medullary thyroid carcinoma in a Brazilian kindred: evaluation of phenotype-modifying effect of germline variants. Clin Endocrinol (Oxf) 67:806-8 Também participo de outros trabalhos como co-autor que estão aceitos para publicação ou submetidos à publicação: Gisele Oler, Cléber P. Camacho, Flávio C. Hojaij, Pedro Michaluart Jr., Gregory J. Riggins and Janete M. Cerutti. Gene Expression Profiling of Papillary Thyroid Carcinoma Identifies Transcripts that Are Correlated with BRAF Mutational Status and Lymph Node Metastasis. Aceito para publicação, Clinical Cancer Research. Helton E. Ramos, Leando A. Diehl, Cléber P. Camacho, Petros Perros, Hans Graf. “Management of Graves’ Orbitopathy in Latin America: an International Questionnaire Study compared with Europe”. Aceito para publicação, Clinical Endocrinology Rosana Tamanaha, Cléber P. Camacho, Alexandre C. Pereira, Adriana M. Álvares da Silva, Rui M.B. Maciel and Janete M. Cerutti. Evaluation of RET polymorphisms in a sixgeneration family with G533C RET mutation: specific RET variants may modulate age at onset and clinical presentation. Submetido à revista The Journal of Clinical Endocrinology and Metabolism. Ricardo A. Guerra, Felipe Crispim, Cláudia C. D. Nakabashi, Cleber P. Camacho, Teresa S. Kasamatsu, Rui M. B. Maciel. "Thyroglobulin is still detectable in the serum of normal individuals after a trial of TSH-suppressive doses of L-T4 during 3 months". Submetido à revista Thyroid iii Helton E. Ramos, Valter T. Boldarine, Suzana Nesi-França, Cleber P. Camacho, Gustavo S. Guimarães, Magnus R. Dias da Silva, Hans Graf, Luiz de Lacerda, Gisah A. Carvalho, Rui M. B. Maciel. "NKX2.5 missense mutations are associated with thyroid ectopy: a study of 157 patients with thyroid dysgenesis". Submetido à revista Journal of Clinical Endocrinology and Metabolism Helton E. Ramos, Suzana Nesi-França, Valter T. Boldarine, Rosana M. Pereira, Maria Izabel Chiamolera, Cleber P. Camacho, Hans Graf, Luiz de Lacerda, Gisah A. Carvalho, Rui M. B. Maciel. "Clinical and Molecular Analysis of Thyroid Hypoplasia: A Population-based Approach in Southern Brazil". Submetido à revista Thyroid. iv INTRODUÇÃO As neoplasias malignas da tiróide são as neoplasias endócrinas mais comuns (90%) e respondem por mais da metade das mortes por cânceres endocrinológicos. O uso da ultra-sonografia na avaliação da doença nodular da tiróide foi de extrema importância ao auxiliar o diagnóstico precoce destas neoplasias, mas ao mesmo tempo gerou um grande problema, já que aumentou a incidência de nódulos tiroidianos para até 67% (6). Este aumento da incidência dos nódulos tiroidianos teve duas conseqüências inevitáveis: o aumento do número de citologias aspirativas com agulha fina (CAAF) com diagnóstico indeterminado e o aumento na incidência da neoplasia maligna da tiróide, basicamente pelo aumento da detecção de carcinomas papilíferos pequenos (7). Até o momento, os diagnósticos indeterminados representam 10 a 20% das CAAF. A incapacidade diagnóstica dos métodos tradicionais, particularmente da CAAF (8), gerou uma corrida dos centros de pesquisa por métodos alternativos que conseguissem aumentar a sensibilidade e especificidade de tal citologia. Mesmo antes desta “epidemia relativa” de cânceres de tiróide, já se pesquisavam os mecanismos responsáveis pela gênese das neoplasias malignas da tiróide (9, 10). Assim, dentre os eventos relacionados com a gênese do câncer tiroidiano, identificou-se o gene RET, que codifica uma proteína cinase transmembrana, a qual, através do rearranjo com outros genes, é relacionada com a gênese do carcinoma papilífero da tiróide. Além disso, a mutação ativadora do RET foi relacionada com a gênese do carcinoma medular da tiróide. Recentemente, identificou-se a mutação V600E no BRAF como sendo o evento responsável pela maioria dos casos de carcinoma papilífero. 1 No entanto, o maior desafio cerca o carcinoma folicular da tiróide (FTC), que apresenta as características de célula tiroidiana diferenciada, mas que não evidencia alterações nucleares características do carcinoma papilífero da tiróide, as quais permitem o diagnóstico deste subtipo através da CAAF. Seu diagnóstico tem sido realizado apenas histologicamente, depois da cirurgia, por meio da identificação de invasão tumoral da cápsula, dos vasos ou do tecido tiroidiano adjacente, o que o diferencia do adenoma folicular da tiróide (FTA), lesão benigna. O FTC pode responder por até 50% dos tumores diferenciados da tiróide, com maior freqüência em regiões carentes de iodo (11). O FTC pode se apresentar sob duas formas: uma minimamente invasiva (MIFC) e a outra extensamente invasiva (WIFC). A MIFC apresenta um prognóstico semelhante ao do FTA, sendo necessária avaliação criteriosa por parte do patologista, para que este subtipo seja corretamente classificado. O WIFC é mais facilmente diferenciado das lesões benignas. Mesmo na forma WIFC, é extremamente rara a ocorrência de metástase para linfonodos cervicais, sendo observadas, em 5 a 20% dos casos, metástases à distância, principalmente para pulmões e ossos. Vários fatores moleculares foram até agora apontados como participantes da patogênese do FTC, mas nenhum deles pode ser considerado como sua causa predisponente principal (10). Assim, mutações ativadoras do oncogene RAS são conhecidas há algum tempo e estão presentes em aproximadamente 40% dos FTC, mas podem ser encontradas também em até 20% do FTA e em 10% dos carcinomas papilíferos da tiróide (12). Kroll e cols. nos levaram a acreditar que o desafio de identificar um evento responsável por esta neoplasia havia chegado ao fim (13). No entanto, o rearranjo do PAX8 e do PPARγ também foi encontrado em neoplasias benignas da tiróide (14). Ying e cols. geraram o primeiro modelo animal de carcinoma folicular da tiróide através de uma mutação no receptor do hormônio tiroidiano (15). 2 Apesar da fisiopatologia do FTC continuar desconhecida, Cerutti e cols. conseguiram identificar 4 novos marcadores: DDIT3, ARG2, ITM1 e Clorf24, que, quando usados em combinação, são capazes de distinguir as lesões adenoma folicular de carcinoma folicular com alta sensibilidade e especificidade (1). Estudo posterior demonstrou que tais marcadores distinguem outras lesões da tiróide comumente classificadas como padrão folicular (16) e, desta forma, representaram um grande passo na questão das CAAF indeterminadas, além de ser uma pista importante na determinação das vias envolvidas na patogênese do carcinoma folicular da tiróide. O tratamento atual dos tumores diferenciados da tiróide é baseado na realização da tiroidectomia total e da radioiodoterapia, sendo eficiente na grande maioria dos casos. No entanto, naqueles pacientes em que estes procedimentos não foram capazes de alcançar a cura, a radioterapia externa e a quimioterapia são os procedimentos alternativos existentes e apresentam resultados variáveis. O lítio e o ácido retinóico são usados como adjuvantes ao radioiodo, sendo observados resultados insatisfatórios. A pesquisa com novas drogas para alvos moleculares é uma nova esperança, mas no caso do FTC, ainda não foram identificados tais alvos (17). Neste contexto, iniciamos a busca por genes diferencialmente expressos que pudessem ser avaliados em modelos experimentais e que tivessem um papel potencial na terapia do câncer, sendo este o enfoque do manuscrito a seguir. 3 OBJETIVOS 1) Validar a expressão de NR4A1 nos tecidos benignos e malignos da tiróide. 2) Avaliar a correlação dos genes CCND1 e FOSB com a expressão de NR4A1. 3) Verificar a expressão dos genes NR4A1, CCDN1 e FOSB nas linhagens de carcinoma tiroidiano disponíveis no laboratório, com objetivo de indetificar um modelo semelhante a expressão observada nos tecidos. 4) Verificar se a expressão dos genes NR4A1, FOSB e CCDN1 poderia ser restabelecida quando tratadas com lítio. 4 Down-regulation of NR4A1 in follicular thyroid carcinomas is restored following lithium treatment Cléber P. Camacho1,2, Flavia R. M. Latini1,2, Gisele Oler1,2, Flavio C. Hojaij3, Rui M. B. Maciel1, Gregory J. Riggins4 and Janete M. Cerutti1,2 1 Genetic Bases of Thyroid Tumors Laboratory, Division of Genetics, Department of Morphology, 2Laboratory of Molecular Endocrinology, Division of Endocrinology, Department of Medicine; and 3Division of Head and Neck Surgery, Federal University of São Paulo, São Paulo, Brazil; 4Departament of Neurosurgery, Johns Hopkins University Medical School, Baltimore, Maryland, USA Correspondence: Janete Cerutti, Ph.D. Genetic Bases of Thyroid Tumor Laboratory Rua Pedro de Toledo 669, 11° andar. Federal University of São Paulo 04039-032, São Paulo, SP, Brazil Phone: +55-11-5081-5233 fax: +55-11-5084-5231 E-mail: [email protected] Running title: NR4A1 expression in thyroid tumors Key words: Follicular Thyroid Carcinoma, NR4A1, CCND1, FOSB and lithium. Acknowledgements: We gratefully acknowledge funding from the São Paulo State Research Foundation (FAPESP) grants 05/60330-8, NIH Grant CA113461, and the Ludwig Center at Johns Hopkins. CPC is a scholar from CAPES, JMC and RMBM are investigators of the Brazilian Research Council (CNPq), GO and FRML are scholars from FAPESP and GJR is the the Irving J. Sherman M.D. Neurosurgery Research Professor. 5 Abstract Introduction: The identification of follicular thyroid adenoma-associated transcripts will lead to a better understanding of the events involved in pathogenesis and progression of follicular tumors. Using Serial Analysis of Gene Expression, we identified five genes that are absent in a malignant follicular thyroid carcinoma (FTC) library, but expressed in follicular adenoma (FTA) and normal thyroid libraries. Methods: NR4A1, one of the five genes, was validated in a set of 27 normal thyroid tissues, 10 FTAs and 14 FTCs and three thyroid carcinoma cell lines by real time PCR. NR4A1 can be transiently increased by a variety of stimuli, including lithium, which is used as adjuvant therapy of thyroid carcinoma with 131 I. We tested if lithium could restore NR4A1 expression. The expression of other genes potentially involved in the same signaling pathway was tested. To this end, lithium was used at different concentration (10mM or 20mM) and time (2h and 24 h) and the level of expression was tested by quantitative PCR. We next tested if Lithium could affect cell growth and apoptosis. Results: We observed that NR4A1 expression was under-expressed in most of the FTCs investigated, compared to expression in normal thyroid tissues and FTAs. We also found a positive correlation between NR4A1 and FOSB gene expression. Lithium induced NR4A1 and FOSB expression, reduced CCDN1 expression, inhibited cell growth and triggered apoptosis in a FTC cell line. Conclusions: NR4A1 is under-expressed in most of FTCs. The loss of expression of both NR4A1 and the Wnt pathway gene FOSB was correlated with malignancy. This is consistent with the hypothesis that its loss of expression is part of the transformation process of FTCs, either as a direct or indirect consequence of Wnt pathway alterations. Lithium restores NR4A1 expression, induces apoptosis and reduces cell growth. These findings may explain a possible molecular mechanism of lithium’s therapeutic action. 6 Introduction The number of thyroid cancer cases is increasing and for women this malignancy has one of the highest rates of increased incidence in the United States. Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are the most frequent thyroid cancer subtypes1 . During the last two decades we have seen progress in our understanding of the molecular biology of these cancers. The importance of the MAPK pathway has been identified in PTC, resulting from RAS/BRAF mutations or rearrangements of the RET oncogene 2-4 . Less is known about the molecular events underlying FTC. The most frequent genetic alterations in FTC comprise point mutations of the RAS genes and PAX8PPARγ rearrangements. Although they were initially proposed as potentially associated with pathogenesis of FTC, these genetic events also occur with significant prevalence in FTA5-9 .Although it has been suggested that those FTAs with PAX8-PPARγ are more likely to transform into a malignant tumor10, 11 , to date there is no compelling in vitro or in vivo evidence. Moreover, a high proportion of FTCs are negative for the rearrangement7 and, therefore, unrevealed genes or pathways may be involved in the pathogenesis of this type of thyroid tumor. The identification of novel FTA-associated transcripts, could lead to a better understanding of the events involved in pathogenesis and progression of FTC. We compared the gene expression profiles of three SAGE libraries generated from thyroid tumors and a normal thyroid tissue12, 13 . The expression analysis was performed originally to locate clinical markers and the data are posted at http://cgap.nci.nih.gov/SAGE. Using SAGE Digital Gene Expression Displayer (DGED), a tool to compare SAGE libraries, we identified five genes that are expressed in normal thyroid and FTA but not expressed in the malignant FTC. One of these genes, NR4A1, encodes an orphan member of the steroid/thyroid/retinoid super-family, whose members act mainly as transcription factors that induce or repress gene expression14 . NR4A1 (Nuclear receptor subfamily 4, group A, member 1) was of particular interest because it was under-expressed in several tumors compared with the corresponding normal tissues15, 16 and because NR4A1 appears to up-regulate pro-apoptosis genes17, 18 . Consequently, one can hypothesized that lost or reduced NR4A1 expression in tumors may be associated with malignant transformation of follicular thyroid cells. Although the mechanisms involved in the 7 transcriptional regulation of NR4A1 is not completely understood, it was described as a target of Wnt-1 (Wingless-type MMTV integration site family, member 116 . The best understood Wnt pathway is the canonical pathway, which controls cell proliferation through inhibition of glycogen synthase kinase-3 beta (GSK3β) and increase in transcriptional activation of β-catenin targets. Although the authors described NR4A1 as a Wnt-1 responsive gene with decreased expression in tumors, they suggested that NR4A1 may be up-regulated by a noncanonical Wnt signaling pathway16 . NR4A1 was also of interest because its expression can be transiently increased by a variety of stimulus, including lithium a drug that has been used to therapy of thyroid cancer16, 19-21 . It was shown that lithium acts as a specific inhibitor of the glycogen synthase kinase-3 beta (GSK3β). Although Lithium has been used as adjuvant to the 131I therapy of thyroid cancer, the cellular target of lithium is still unknown. We investigated the expression of NRA41 in a set of normal thyroid tissues, FTAs and FTCs. Subsequently, we investigated the level of expression of other genes downstream the Wnt signaling pathway and correlate their expression with NR4A1 expression. We next sought to test whether lithium could restore the NR4A1 expression in thyroid cell lines and explain a possible molecular mechanism for the effect of lithium as an adjuvant therapy for thyroid cancer. 8 Material and Methods SAGE Digital Gene Expression Displayer. DGED was used to identify genes differentially expressed between benign (normal tissue and FTA) and malignant (FTC) SAGE libraries. The full set of tag counts for all thyroid libraries is available for downloading or analysis at the Cancer Genome Anatomy Project SAGE Genie Web site (http://cgap.nci.nih.gov/SAGE)12 . To identify genes differentially expressed, we defined as criteria P <0.05 and 10-fold difference in the level of expression between the two groups (Fig. 1). Tissue Specimens. A total of 41 thyroid tissue specimens obtained from patients undergoing thyroid surgery for thyroid disease at Hospital São Paulo, Federal University of São Paulo, Brazil, were used in this study. Samples were frozen immediately after surgery and stored at -80°C and included 27 normal thyroids (NTs) 10 FTAs and 14 malignant (FTCs) tissues. Among 14 FTCs, 6 were classified as widely invasive follicular thyroid tumors (WIFC) (capsular and vascular invasion and extension of the tumor beyond the thyroid gland). All tissue samples were obtained with informed consent according to established Human Studies Protocols at Federal University of São Paulo. The study of patient materials was conducted according to the principles expressed in the Declaration of Helsinki. Cell Lines. A FTC (WRO), PTC (NPA) and an undifferentiated thyroid carcinoma (ARO) cell lines used in this study were previously characterized22-25 . Cell lines were grown in DMEM (Invitrogen Corp., Carlsbad, CA) supplemented with 10% FBS (Invitrogen), with 100 units/mL of penicillin and 100 µg/mL streptomycin in a humidified incubator containing 5% CO2 at 37ºC as described26, 27 . RNA extraction and cDNA synthesis and quantitative PCR (qPCR). Total RNA isolation and cDNA synthesis were performed as previously reported13 . Briefly, one microgram of total RNA was treated with a DNAse (Ambion, Austin, TX) and was reverse-transcribed to cDNA using Super-Script II Reverse Transcriptase kit with an oligo(dT)12-18 primer and 10 units of RNase inhibitor (Invitrogen). An aliquot of cDNA was used in 20µL PCR reaction containing SYBR Green PCR Master Mix and 200 nM of each primer for the target (NR4A1, CCDN1 and FOS) or reference genes (ribosomal protein S8 and QPC). Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The PCR reaction was performed for 40 9 cycles of a 4-step program: 94oC for 15 sec, annealing temperature for 20 sec, 72oC for 20 sec, and a fluorescence-read step for 20 sec. qPCR reactions were performed in triplicate. The threshold cycle (Ct) was obtained using Applied Biosystem software and was averaged (SD ≤1). Gene expression was normalized using the average of 2-reference genes. Fold changes were calculated as previously described28 . NR4A1, FOSB and CCDN1 Expression after Lithium Treatment. 5 × 106 cells WRO cells were plated in 35mm plates. After 24 hours, cells were harvest with fresh medium containing lithium chloride (LiCl; Sigma-Aldrich Corp. St. Louis, MO, USA). Concentration (10mM and 20mM) and time (2 and 24 hours) were determined according to previous studies16, 29 . Doses of 10mM and 20mM lithium are commonly used to achieve GSK3β inhibition and activation of Wnt/β-catenin signaling29. Total RNA isolation, cDNA synthesis and qPCR for NR4A1, FOSB, CCDN1 and reference genes were performed as described above. Additionally, the SodiumIodide-Symporter (NIS) expression was tested following lithium treatment. MTT Assay. We next investigated the effect of lithium on cell proliferation. In brief, 2 x 104 WRO cells were seeded in 35-mm in quintuplicate, allowed to adhere overnight, and then treated with lithium at 10mM and 20mM concentrations. At each time point, cell growth rates were analyzed following addition of 3,4-(4,5dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) (Sigma) reagent to the cultured cells as described30 . Absorbance was measured using spectrophotometer at a wavelength of 560 nm. Apoptosis Assay. We investigated if lithium could induce apoptosis in WRO cells. Briefly, 2 × 104 cells WRO cells were plated in 35mm plates in quintuplicate. Cells were then treated with lithium at 10mM and 20mM concentrations. At indicated time, Annexin V–positive cells were measured using the Guava Nexin kit as described30 . Statistical Analysis. qPCR values were log-transformed before application of statistical tests. Unpaired student t test was used to examine differences between two groups and ANOVA with Bonferroni correction was used to examine differences among groups (StatView software). To test the significance of the relationship between genes, Pearson correlation was used. Chi-squared (X2) with odds ratio (OR) was performed to determine the risk for tumorigenesis. A P value of < 0.05 was considered significant. 10 Results Genes Potentially Associated with Pathogenesis of Thyroid tumors. Differentially expressed genes can be found by comparison of various SAGE libraries using the SAGE Digital Gene Expression Display (SAGE DGED)31 . We compared two pools of thyroid SAGE libraries. Pool A included both normal thyroid and FTA and pool B consisted of FTC library. Using P <0.05 as the value for statistical significance and 10-fold as the difference in the level of expression, we identified 6 transcripts differentially expressed between pool A and pool B. Our primary focus was on genes whose expression was lower in FTC library such as TPO, TFF3, NR4A1, and IYD (Fig. 1). NR4A1 was selected for further investigation. NR4A1 Expression in Thyroid Tissues. The data predicted by SAGE was validated by qPCR using a set of 27 normal thyroid tissues, 10 FTAs and 14 FTCs. We observed that NR4A1 expression was under-expressed in most FTCs investigated (65%), compared with the expression observed in normals (P=0.01) and FTAs (Fig. 2A). The expression was remarkably lower in widely invasive follicular carcinoma than adenoma (P=0.02) (Fig. 2B). To determine the risk of tumorigenesis, the level of NR4A1 expression was analyzed in FTCs (n=14) and normal thyroid (n=22) groups. Using 0.1 as the cutoff value, expression levels lower than 0.1 were observed in 5 of 22 normal thyroids and 8 of 14 FTCs. A low level of NR4A1 expression was associated with an increased risk of FTC (Odds ratio= 5.8; χ2:6,352; P = 0.0117; IC95%: 1.39 – 24.67). To provide a more accurate estimate of the risks of malignancy associated with NR4A1 level of expression, a larger series and absolute values are needed. FOSB and CCND1expression in thyroid Samples. Since NR4A1 was suggested as downstream of Wnt-1 signaling pathway, we investigated the expression of canonical Wnt-1 target genes such as FOSB and CCDN1. Additionally, we sought to determine whether there is a correlation among expression levels for NR4A1, FOSB and CCND1. FOSB was under-expressed in most of thyroid carcinomas and a number of FTAs compared to normal thyroid, although not significant (Fig. 2C). Intriguingly, CCDN1 was over-expressed in both FTCs (P < 0.01) and FTAs (P = 0.01), compared to normal thyroid (Fig. 2D). 11 We found a positive correlation between NR4A1 and FOSB genes in normal tissue (P= 0.0065), adenoma (P= 0.0429) and carcinoma tissue (P= 0.0020) (Fig. 2E). There was no correlation between NR4A1 and CCND1 (Fig. 2F). Lithium restores NR4A1 and FOSB expression and reduced CCND1 expression in FTC cell line. We next investigated expression levels of NR4A1, CCND1 and FOSB in thyroid carcinoma cell lines: NPA, ARO and WRO. NR4A1 and FOSB were under-expressed in all cell lines compared to normal thyroid. CCND1 was overexpressed in WRO, compared to normal thyroid (Fig. 3A, B and C). Since the WRO follicular thyroid carcinoma cell line had patterns expression of the tested genes similar to those observed in FTCs, it was selected for further analysis. WRO cells were cultured in medium containing Lithium Chloride (LiCL) at different concentrations and time and the level of expression of selected genes were determined. In multiple experiments, qPCR analysis showed a consistent increased of NR4A1 transcripts in 2h lithium-treated cells compared with untreated controls, in a dose-dependent manner. A greater effect was observed when cells were treated with 20 mM LiCl for 2 h (Fig 4A). After 24 h, NR4A1 expression was up-regulated only at high doses of lithium (Fig 4B). The expression of FOSB, an early gene, was restored after 2 h treatment when 20 mM lithium was used and was down-regulated in cells exposed to 10 mM LiCl for 2 h (Fig. 4C and D). FOSB was down-regulated in and 24 h lithium-treated cells compared with untreated controls, in a dose-dependent manner (Fig. 4D). The expression of CCND1 was biphasic with a slight increase at 2 h of lithium treatment followed by a down-regulation at 24 h as compared with untreated control cells. Comparable effects of lithium on CCND1 expression were previously reported32 (Fig. 4E and 4F). Since lithium has been suggested to act as an adjuvant to 131 I therapy by inhibiting its release from the thyroid, we tested whether the expression of the sodium-iodide symporter (NIS) changed after lithium treatment. We did not find any difference in expression of NIS in WRO-treated cells compared with non-treated controls (data not shown). 12 Lithium inhibits cell growth and induces apoptosis in FTC cell line. Although controversy, previous studies showed that over-expression of NR4A1 inhibited cell growth and induced apoptosis. Since lithium restores NR4A1 expression in FTC cell line, we investigated the effect of lithium on cell growth and apoptosis. As showed in Fig. 5A, lithium treatment resulted in a progressive and dose-dependant cell growth inhibition as compared with the untreated control cells. The reduction in cell proliferation in 20 mM lithium treated cells was statistically significant in all treatment points compared to control cells (P<0.001). To quantify the apoptotic rate of WRO cells following treatment, different concentration of lithium were tested at 48 and 72 hours, a time in which growth inhibition was more evident for both concentrations. Lithium significantly increased the number of apoptotic cells in a time and dose-dependent manner (P<0.001) (Fig. 5B). 13 Discussion In this study, using SAGE Digital Gene Expression Display and validation by qPCR, we found that NR4A1 was under-expressed in most of FTCs compared to normal thyroid and FTA, and markedly lower in widely invasive follicular thyroid carcinoma. NR4A1, also known as Nur77, TR3 and NGFI-B, an orphan nuclear receptor, was first described as an immediate early gene induced in response to serum or phorbol ester stimulation of quiescent fibroblasts. Although it was initially known for its role in cell proliferation and differentiation, later was shown to be a potent pro-apoptotic molecule in different cell types. Inhibition of NR4A1 activity either by over-expressing a dominant-negative form or using antisense RNA, inhibited apoptosis33, 34 . Consistent with its apoptotic activity, over-expression of NR4A1 in some cell lines leads to apoptosis. It was demonstrated that its apoptotic activity can be altered through AKT-mediated phosphorylation in the DNA binding domain of NR4A1 and substantially diminishes its ability to bind DNA and diminish its apoptotic activity35 . Others have suggested that NR4A1 is phosphorylated through the ERK5 pathway36 . Moreover, it was suggested that NR4A1-mediated apoptosis involves transcription-independent mitochondrial targeting of NR4A1. When induced by apoptotic stimuli, NR4A1 translocates into the cytoplasm where it binds to Bcl2 and provokes conformational change of Bcl2. This results in conversion of Bcl2 from a cell protector to a cell killer37, 38 . Based on its apoptotic effects, many studies were performed searching for a link between tumorigenesis and NR4A1 expression. NR4A1 expression was reduced in several tumor subtypes compared to normal matched samples16 . Recently it was described that down-regulation of NR4A1 was a common feature in leukemia blast cells from human acute myeloid leukemia (AML)39 . Additional evidence that supports a role for NR4A1 in tumorigenesis comes from the observation that NR4A1 abrogation in mice led to a rapidly lethal and transplantable leukemic disease with pathological features most closely resembling AML. Based on these findings, it was suggested a new role of NR4A1, a critical tumor suppressor of myeloid leukemogenesis39 . 14 The aforementioned evidences of its potential role as a tumor suppressor gene and the fact that we found a decreased expression of NR4A1 in follicular thyroid carcinomas compared to normal thyroid and FTA, suggest that the down-regulation of NR4A1 expression might be an important event during thyroid tumorigenesis. Given that NR4A1 induction is regulated by a variety of cell- and stimulus-specific such as growth factors, calcium, inflammatory cytokines, peptide hormones, phorbol esters, neurotransmitters and lithium16, 19, 40, we hypothesize whether these agents could restore the expression of NR4A1. The effect of lithium on thyroid was first observed in patients who received lithium for mood diseases and developed goiter41 . Later it was demonstrated that lithium inhibits the release of radioiodine from the thyroid42 . Due to this effect lithium is considered an option for adjuvant treatment with radioiodine for selected cases of thyroid cancers21, 43, 44 . Based on the fact that lithium has been used as therapeutic drug for thyroid carcinomas for several years but it therapeutic action remains an enigma and that it induces NR4A1 expression16 , we hypothesize that lithium might exert its therapeutic effect by restoring the expression of NR4A1. We show here that when a follicular thyroid carcinoma cell was exposed to 10 mM or 20 mM of lithium, NR4A1 expression increased at significant levels after 2 hours of treatment, in a dose-dependent manner. Additionally, since NR4A1 over-expression in response to a variety of stimuli and chemotherapeutic agents was involved in growth suppression and apoptosis of many cell types18, 45 , we further examined if lithium could trigger apoptosis and reduce cell growth in a FTC cell line. We demonstrated here that lithium inhibited cell growth and induced apoptosis in a dose dependent manner. This helps implicate a potential role for NR4A1 in thyroid cells and therapeutic mechanism of lithium action. However, we do not know yet if induction of the cell death pathway by lithium is accompanied by translocation of NR4A1 from the nucleus to the cytosol/mitochondria or by post-trascriptional modification by ERK5 or by AKT-mediated phosphorylation. These possibilities need further investigation. Converse to our observations, it has been reported that 5 mM lithium stimulates proliferation of two thyroid carcinoma cell lines (papillary and follicular). The authors suggested that inhibition of GSK3β by lithium 15 lead to an enhancement of proliferation by increasing the β-catenin levels29 and, therefore, mimic the canonical Wnt/β-catenin signaling 29,46 . However, it was demonstrated that although the inhibition of the GSK3β pathway occurred at concentrations as low as 1 mM lithium, substantial growth inhibition of tumor cells does not appear until treatment with 10 mM lithium47 . Recently, it was demonstrated that treatment with lithium inhibited growth of a medullary thyroid carcinoma cell line (TT) both in vivo and in vitro and that exerts its effect via inhibition of the glycogen synthase kinase 3β (GSK3β). Increased concentration of lithium significantly reduced growth of TT cells in a dose dependent fashion48 . Inhibition of GSK3β by specific inhibitors has shown to reduce tumors growth in other cancers including colon, melanoma and glioblastoma49-51. Therefore, these conflicting results could be explained not only for the different concentrations used but also by the fact that the effect of lithium may not be general but cell type specific. Since it was described that lithium is able to mimic Wnt signaling46 it was logical to assume that lithium could modulate other genes downstream in the canonical Wnt/β-catenin signaling pathway. We therefore, investigated whether lithium modulated the expression of previously described Wnt-1 target genes CCND152 and FOSB53 . We observed a rapid and dose-dependent increased expression of FOSB in thyroid cells following lithium treatment. The rapid and transient expression of FOSB parallel those observed for NR4A1, suggested they may have a similar mechanism of activation. Our results are in accordance with the literature where a reduced expression of the immediate early gene FOSB was observed following stimulation of cells by Wnt-153 . This study also demonstrated that the expression of FOSB was significant reduced in human colon cancer relative to normal tissue53 . We therefore investigated the expression of FOSB in the set of samples where NR4A1 expression was tested. We not only found a reduced expression of FOSB in thyroid tumor compared to normal thyroid but a positive correlation with NR4A1. CCND1, a reduced expression of CCND1 was observed after 24 h treatment with lithium. Kunnimalaiyaan et al.,48 previously demonstrated a decreased expression of CCND1 parallel to the increased 16 expression of cell cycle inhibitors such as p21, p15 and p27 following lithium treatment. Whether lithium inhibits GSK3β and restores NR4A1 and FOSB expression and decreases CCND1 expression through the canonical Wnt/β-catenin signaling pathway, is still not clear. Although CCND1 was over-expressed in follicular tumors, there was no correlation with the expression of NR4A1. Of note, CCND1 over-expression and FOSB under-expression were previously described as associated with tumorigenesis of papillary thyroid carcinoma54, 55 . Whether NR4A1 play a role in the pathogenesis of papillary thyroid carcinoma (PTC) is controversy. Previous studies using gene expression profiling following expression of genetic events that are hallmarks of PTC demonstrated that NR4A1 was among the genes found under-expressed in cells expressing either RET/PTC3, RAS or BRAF genetic alteration, although not confirmed by qPCR56 . Conflicting evidence is that NR4A1 was described as one of the genes induced by RET/PTC357 . In conclusion, we show that NR4A1 is down-regulated in follicular thyroid carcinoma compared to normal thyroid and follicular adenomas. This event may be associated with cancer development. 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(2003) Ret/PTC activation does not influence clinical and pathological features of adult papillary thyroid carcinomas. Eur J Endocrinol, 148,505-13. 24 Figure Legends Fig. 1. SAGE Digital Gene Expression Displayer (DGED). The comparison of thyroid SAGE libraries identified those genes differentially expressed genes greater than 10-fold with P<0.05. Pool A includes a normal thyroid library and a follicular thyroid adenoma library. Pool B includes a follicular thyroid carcinoma library. Fig. 2. Relative expression (RE) analysis of selected genes in thyroid tumors and normal thyroid. Transcripts levels were normalized to the average of 2-internal controls. The qPCR data were log transformed for statistical analysis. Changes were calculated by comparison of the mean expression value. (A) NR4A1 was down-regulated in follicular thyroid carcinoma compared to normal thyroid (P=0.01). (B) The expression of NR4A1 was dramatically reduced in widely invasive thyroid carcinoma (WITC) compared to adenoma (P=0.02). (C) Although FOSB was down-regulated in follicular carcinomas compared to normal thyroid, it was not considered significant (D) CCND1 was over-expressed in both follicular adenoma (P=0.01) and follicular carcinoma(P=0.006) compared to normal thyroid. (E) All cases together showing a positive correlation was found between the levels of NR4A1 and FOSB. (F) All cases together, showing no correlation between NR4A1 and CCND1. Expression was significantly different at the 0.05 level (*). Fig. 3. Relative levels of expression of NR4A1 (A), FOSB (B) and CCND1 (C) determined by qPCR in a papillary thyroid carcinoma cell line (NPA), follicular thyroid carcinoma cell line (WRO) and undifferentiated thyroid carcinoma cell line (ARO), compared to the expression (mean) observed in normal thyroid. Fig. 4 Effect of lithium treatment on NR4A1 (A,B) FOSB (C,D) and CCND1 (E,F) expression. A follicular thyroid carcinoma cell line (WRO) was treated with LiCl 10mM (L10) and 20mM (L20) for 2 hours and 24 hours. Transcripts levels were normalized to the average of 2-internal control and qPCR data are averaged from two experiments performed in triplicate. The qPCR data were log transformed for statistical analysis. Changes were calculated by comparison of the mean expression value. Expression was considered significant when P<0.05 (*) and P<0.01(**). Fig. 5 Effect of lithium on cell growth (A) and apoptosis (B) in a follicular thyroid cell line (WRO). The effect was tested using 10mM (L10) and 20mM (L20). (A) Growth assay by MTT was performed as described in material and methods. Lithium significantly reduced cell growth in a dose-dependent manner. The data represents the mean ± SD of absorbance at 560 nm of experiments performed in quintuplicate on each of four days. (B) To quantify the apoptotic rate of WRO cells following lithium treatment, we analyzed annexin-V binding using a Guava Nexin kit as described in material an methods section. The data represents the mean ± SD of percentage of apoptotic cells following lithium treatment. An increase of apoptosis was observed in a time and dose-dependent manner. Changes were significant when P<0.05 (*) and P<0.001(**). 25 Figure 1 26 Figure 2. 27 Figure 3 28 Figure 4 29 Figura 5 30 CONCLUSÕES Este trabalho apresentou a expressão diferencial do gene NR4A1 entre tecidos benignos e malignos da tiróide, discutindo sua participação na tumorigênese tiroidiana. Foi demonstrada a maior associação do NR4A1 com a forma extensamente invasiva de carcinoma folicular da tiróide, sugerindo que a perda da sua expressão tenha também relação com um maior grau de agressividade. Conseguimos demonstrar uma correlação positiva entre o NR4A1 e FOSB, o que sugere uma via de sinalização comum. No entanto, nenhuma correlação foi observada entre o NR4A1 e CCND1 nos tecidos humanos. Identificamos a linhagem de células WRO como um modelo experimental com o mesmo padrão de expressão observado nos tecidos de carcinoma folicular da tiróide, tanto para o NR4A1 quanto para FOSB e CCND1. A utilização do lítio conseguiu aumentar a expressão do NR4A1, e como esperado também aumentou a do FOSB, mas também reduziu a expressão do CCND1. O lítio foi capaz de alterar o padrão de expressão dos 3 genes nos tecidos benignos da tiróide. O melhor conhecimento do papel do NR4A1 na tumorigênese, assim como das ações do lítio na neoplasia folicular tiroidiana, pode resultar em terapias mais eficazes, para o tratamento dos casos onde não houve resposta ao tratamento convencional com cirurgia e radioiodo. 31 BIBLIOGRAFIA 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 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