Fan, Dahua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 117-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Although there is strong evidence that thyroid tumors occur more frequently in females than in males, few studies have investigated the role sex hormones play in thyroid carcinogenesis, especially the role of estrogen (E2). This laboratory has previously shown that estrogen receptors (ERs) exist in thyroid papillary carcinoma cells. Continuing along this line of research, we studied the role of E2 and its receptors on the regulation of human thyroid cancer. / In conclusion, we have demonstrated (1) a novel mechanism by which E2 contributes to the proliferation and growth of thyroid cancer cells, (2) that E2 influences the expression of ERalpha and ERbeta differently, causing an imbalance between them, which may change the biological behavior of thyroid cancer cells, giving them the ability to proliferate and resist apoptosis by influencing the level of ERK1/2 activity and subsequently the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax, and (3) that the subcellular localization of ERalpha and ERbeta may be a factor that contributes to the differing pathogeneses of papillary and anaplastic thyroid cancers. / To further clarify the mechanism by which E2 promotes cellular proliferation in thyroid cancer cells, we studied the localization of ERalpha and ERbeta in both KAT5 and anaplastic carcinoma cells (FRO) by immunofluorescence staining and by immunoblotting of the proteins in subcellular fractions. Cell proliferation and apoptosis were examined together with the expression of selected apoptotic proteins such as Bax, AIF and cytochrome c. We showed that the subcellular localization of ERalpha and ERbeta differed in papillary and anaplastic thyroid cancer. E2 administration led to an increase in the level of ERalpha in the nuclei of papillary cancer cells while the levels of ERbeta remained unchanged. However, the level of mitochondrial ERbeta surpassed that of ERalpha in anaplastic cancer cells. We also showed that E2 affected caspase-dependent and/or independent apoptosis via ERs in thyroid cancers. / We first studied the molecular pathways by which E2 promotes cellular proliferation in thyroid cancer cells using a human thyroid cancer cell line (KAT5) treated with E2, a selective E2 alpha receptor (ERalpha) agonist (PPT), a selective E2 beta receptor (ERbeta) agonist (DPN), an ERalpha antagonist (MPP), an E2 antagonist (ICI182780) and siRNA, which blocks ERalpha and ERbeta, by MTT assay, DNA fragmentation ELISA, BrdU cell proliferation assay and Western blot. We found that E2 and PPT gradually promoted cell proliferation by increasing the expression of ERalpha and by up-regulating the expression of Bcl-2 and pERK1/2. In contrast, we found that DPN had a negative effect on cell growth by enhancing the expression of ERbeta and Bax and by down-regulating pERK1/2 expression. At the same time, blocking ERalpha significantly reduced the E2-mediated Bcl-2 and pERK1/2 expression. On the other hand, blocking ERbeta markedly enhanced their expression. These results suggest that E2 regulates cellular growth of KAT5 cells by an ER-ERK1/2-MAPK pathway and also that E2 affects mitochondrial homeostasis. / Zeng, Qiang. / "September 2007." / Adviser: George Gong Chen. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4616. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 136-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-06-21T19:17:07Z No. of bitstreams: 1 tairinezaralopes_dissert.pdf: 1511634 bytes, checksum: 2203122e0b39cf1687115059c3e19447 (MD5) / Made available in DSpace on 2016-06-21T19:17:07Z (GMT). No. of bitstreams: 1 tairinezaralopes_dissert.pdf: 1511634 bytes, checksum: 2203122e0b39cf1687115059c3e19447 (MD5) Previous issue date: 2015-10-29 / Introduction: Thyroid cancer is the most common malignancy of the endocrine system and has been presenting continuous increase in the last years. Studies suggest that folate deficiency in the body decrease DNA repair, resulting in malignant cells changes that alter expression of genes, and may induce several kinds of cancer development. Polymorphisms in genes involved in folate pathway have been investigated as risk factors for susceptibility to cancer, among them MTHFR, MTR, RFC1 and CßS. Objectives: To investigate association of polymorphisms in the MTHFR (C677T), MTR (A2756G), RFC1 (A80G) and CßS (844ins68) genes in risk thyroid cancer in a case-control study; to evaluate the association of polymorphisms with gender, age, alcohol and tobacco consumption, body-mass index in thyroid cancer development; and to evaluated the association between polymorphisms and clinical-histopathological parameters. Methods: This study included 462 individuals (151 patients with thyroid cancer and 311 controls). The peripheral blood was collected and genomic DNA was extracted. The MTHFR (C677T), MTR (A2756G) and RFC1 (A80G) were evaluated by PCR-RFLP and CßS (844ins68) by conventional PCR without enzymatic digestion. For statistical analysis chi-square and multiple logistic regression were used. Results: The results showed that MTHFR C677T (OR=2.87, 95% CI=1.50-5.48, p< 0.01, codominant model), (OR=1.76, 95% CI=1.18-2.64, p< 0.01, dominant model), (OR=2.37, 95% CI=1.28-4.39, p< 0.01, recessive model) and RFC1 A80G (OR: 1.55; 95% CI: 1.02-2.38; p=0.04, recessive model) were associated with thyroid cancer. The alcohol (OR=1.56, 95% CI=1.36-1.89, p< 0.01) and tobacco consumption (OR=1.97, 95% CI=1.28-3.04, p< 0.01) were statistically significant, being associated with increased risk. The MTR A2756G is associated with tumor extension (OR=2.69, 95% CI=1.27-5.71, p< 0.01) and aggressiveness (OR= 4.51, 95% CI=1.67-12.1, p< 0.01). Conclusions: The MTHFR (C677T) and RFC1 (A80G) polymorphisms were involved in risk for thyroid cancer. Additionally, alcohol and tobacco consumption increase risk for disease development. / Introdução: O câncer de tireoide é a neoplasia maligna mais comum do sistema endócrino e vem apresentando contínuo aumento nos últimos anos. Estudos sugerem que a deficiência de folato no organismo diminui a reparação do DNA, resultando em alterações celulares malignas que modulam a expressão gênica, podendo levar ao desenvolvimento de vários tipos de câncer. Polimorfismos em genes envolvidos na via do folato têm sido investigados como fatores de risco para suscetibilidade ao câncer, entre eles, polimorfismos nos genes MTHFR, MTR, RFC1 e CßS. Objetivos: Investigar a associação dos polimorfismos nos genes MTHFR (C677T), MTR (A2756G), RFC1 (A80G) e CßS (844ins68) no risco de câncer de tireoide em um estudo caso-controle; Avaliar a associação dos polimorfismos com o gênero, idade, consumo de álcool e tabaco, índice de massa corpórea (IMC) no desenvolvimento do câncer de tireoide; Avaliar a associação entre os polimorfismos e os parâmetros clínico-histopatológicos do câncer de tireoide. Casuística e Método: Este estudo incluiu 462 indivíduos (151 pacientes com câncer de tireoide e 311 indivíduos controles). Foi coletado sangue periférico e extraído o DNA genômico. Os polimorfismos MTHFR (C677T), MTR (A2756G) e RFC1 (A80G) foram avaliados por meio da PCR-RFLP e o polimorfismo CßS (844ins68) foi analisado por PCR convencional sem corte enzimático. Para análise estatística utilizou-se o teste do qui-quadrado e regressão logística múltipla. Resultados: Os resultados mostraram que os polimorfismos MTHFR C677T (OR=2.87, 95% IC=1.50-5.48, p< 0.01, modelo codominante), (OR=1.76, 95% IC=1.18-2.64, p< 0.01, modelo dominante), (OR=2.37, 95% IC=1.28-4.39, p< 0.01, modelo recessivo) e RFC1 A80G (OR: 1.55; 95% IC: 1.02-2.38; p=0.04, modelo recessivo) estão associados ao câncer de tireoide. O consumo de álcool (OR=1.56, 95% IC=1.36-1.89, p< 0.01) e tabaco (OR=1.97, 95% IC=1.28-3.04, p< 0.01) foram estatisticamente significantes, sendo associados ao aumento do risco. O polimorfismo MTR A2756G está associado à extensão do tumor (OR=2.69, 95% IC=1.27-5.71, p< 0.01) e à agressividade (OR= 4.51, 95% IC=1.67-12.1, p< 0.01). Conclusões: Os polimorfismos MTHFR (C677T) e RFC1 (A80G) estão envolvidos no risco de câncer de tireoide. Adicionalmente, o consumo de álcool e tabaco aumenta o risco de desenvolvimento da doença.

Alterações genéticas nos genes RET, RAS e BRAF resultam na ativação constitutiva da sinalização MAPK e estão presentes em aproximadamente 70% dos carcinomas papilífero de tiroide, a forma mais prevalente de câncer de tiroide. Múltiplas vias de sinalização podem atuar em conjunto com a via MAPK na oncogênese tiroidiana. Nesse estudo, testamos a hipótese que a via MAPK regula a sinalização Notch e que o crosstalk entre as vias de sinalizações são importantes na regulação da diferenciação e proliferação celular no câncer de tiroide. A ativação condicional dos oncogenes RET/PTC3 e BRAFT1799A em linhagem de célula folicular normal de tiroide aumentou a atividade da via de sinalização Notch. Por outro lado, o bloqueio farmacológico da sinalização MAPK reduziu a sinalização Notch na linhagem celular TPC-1 derivada de carcinoma papilífero de tiroide. Glândulas tiroide de animais transgênicos expressando BRAFT1799A e amostras de carcinoma papilífero de tiroide apresentaram elevados níveis de NOTCH1. A superexpressão de NOTCH1 em célula folicular normal de tiroide aumentou a expressão proteica de NIS. A inibição farmacológica e por RNA de interferência da sinalização Notch apresentou um efeito anti-proliferativo em linhagem de CPT. Além disso, a combinação do inibidor farmacológico de Notch e MAPK diminuiu a proliferação de células de carcinoma papilífero de tiroide. Esses dados sugerem um importante papel da sinalização Notch na oncogênese do carcinoma papilífero de tiroide induzida pela sinalização MAPK e que a via Notch pode ser uma potencial terapia adjuvante no câncer de tiroide. / Genetic alterations in RET, RAS and BRAF result in constitutive activation of the MAPK signaling and are present in approximately 70% of papillary thyroid carcinomas, the most prevalent form of thyroid cancer. Multiple signaling pathways can act with MAPK pathway in thyroid oncogenesis. In this study, we tested the hypothesis that MAPK pathway control Notch signaling and that the crosstalk between these pathways plays an important role in thyroid cancer cell differentiation and proliferation. The conditional activation of RET/PTC3 and BRAFT1799A enhanced Notch signaling pathway in normal follicular thyroid cell. By contrast, pharmacological inhibition of MAPK reduced Notch signaling in TPC-1 cell line derived from papillary thyroid carcinoma. Transgenic mice expressing BRAFT1799A restrict in thyroid gland and human papillary thyroid carcinoma samples showed higher Notch1 expression. NOTCH1 overexpression in normal thyroid follicular cell increased NIS protein expression. Pharmacological inhibition and RNA interference of Notch signaling showed an anti-proliferative effect in papillary thyroid carcinoma cells. Furthermore, the combination of MAPK and Notch signaling inhibitors reduced papillary thyroid carcinoma proliferation. These data suggest an important role of Notch signaling in papillary thyroid carcinoma induced by MAPK-related oncogenes and that Notch signaling pathway could be a potential adjuvant therapy in thyroid cancer.

O câncer da tireoide é a neoplasia endócrina mais comum, sendo responsável por cerca de 1 a 2% das neoplasias malignas da tireoide. Atualmente, a patogênese molecular do carcinoma papilífero da tireoide (CPT) tem sido relacionada à ativação aberrante da via de sinalização MAPK, desencadeada por mutações em diversos oncogenes. Destas, a mutação p.V600E do gene BRAF é a mais freqüente, sendo observada em 30%-80% dos casos. Numerosos estudos têm demonstrado que a presença dessa mutação está relacionada a uma maior agressividade do tumor e, conseqüentemente, a um prognóstico menos favorável, tornando-a um marcado importante no CPT. Contudo, poucos métodos utilizados na análise do gene BRAF em amostras de punção de nódulos tireoidianos foram satisfatórios em relação ao custo-tempo e sensibilidade do teste. Os objetivos deste estudo foram padronizar a extração de DNA a partir de amostras obtidas de PAAF guiada por ultrassom de nódulos tireoidianos; validar e determinar a eficiência e a relação custo-tempo da técnica de genotipagem por PCR em tempo real na detecção da mutação p.V600E do gene BRAF em amostras de PAAF de nódulos tireoidianos; analisar a prevalência da mutação p.V600E em pacientes com CPT; correlacionar à presença da mutação p.V600E com características clínicas e histopatológicas de maior agressividade e por fim analisar a sensibilidade, especificidade e acurácia do diagnóstico citológico em conjunto com a análise molecular da mutação p.V600E em material de PAAF. Nossa casuística foi composta por 224 pacientes, todos submetidos à tireoidectomia, cuja citologia pré-operatória foi indeterminada (Bethesda classes III a V) ou Bethesda VI (carcinoma papilífero). Foram avaliados dados clínicos e hormonais (TSH) e autoimunidade (anti-TPO e anti-TG), além das características ultrassonográficas (tamanho, estrutura, ecogenicidade, presença de microcalcificação e halo e vascularização). Os dados histológicos avaliados foram tamanho do tumor, variante histológica, invasão de cápsula, invasão vascular e linfática, extensão extratireoidiana, multicentricidade e presença de acometimento ganglionar à cirurgia. Os pacientes foram divididos em grupo benigno (n:122) e maligno (n:102), de acordo com o diagnóstico histológico final. O grupo maligno apresentou uma média de idade menor que o grupo benigno (48,9 vs 54,2 anos; p=0,008). Não observamos diferença entre os grupos com relação à dosagem sérica de TSH (p=0,467), anti-TPO (p=0,535) e anti-TG (p=0,730). Analisando as características ultrassonográficas, o tamanho e o volume dos nódulos foram maiores no grupo benigno (3,0 vs 2,6 cm e 12,38 vs 14,5 cm3; p=0,008 e p<0,001, respectivamente). Os nódulos que apresentaram hipoecogenicidade, assim como os nódulos de composição sólida, a presença de microcalcificações, ausência de halo hipoecogênico e presença de vascularização central apresentaram estatisticamente maior freqüência de malignidade. Em modelo de regressão logística, maior idade, nódulo sólido, sem halo hipoecogênico e com microcalcificações foram variáveis que influenciaram conjuntamente na presença de malignidade. No diagnóstico citológico 78,6% (176/224) dos nódulos avaliados foram classe III, IV e V, sendo que destes 35,8% (63/176) apresentaram diagnóstico histológico final maligno. Identificamos a mutação p.V600E no material de PAAF em 67,7% (69/102) dos pacientes do grupo maligno, estando presente em 70,3% (45/64) dos carcinomas papiliferos variante clássica e em 69,7% (23/33) dos carcinomas papilíferos variante folicular, sendo todos os achados confirmados em 100% das amostras através seqüenciamento automático do material obtido do tecido nodular a fresco ou parafinado dos pacientes. Nos pacientes com diagnóstico histológico de CPT (n:98), comparamos os pacientes com e sem a mutação p.V600E com relação aos dados clínicos, histológicos de pior prognóstico, presença de metástase linfonodal, classificação TNM e estádio de acordo com AJCC. Apenas a presença de idade mais avançada apresentou associação estatisticamente significativa com a presença da mutação p.V600E (p=0,041). Comparamos o diagnóstico citológico baseado na classificação de Bethesda e a análise da mutação p.V600E com o diagnóstico histológico considerado o padrão ouro para o diagnóstico de CPT. A sensibilidade, especificidade, acurácia, valor preditivo positivo e negativo do diagnóstico citológico foi de 67,4%, 94,4%, 79,8%, 93,3% e 71,2% respectivamente. Análise da mutação p.V600E isoladamente apresentou resultados similares ao do diagnóstico citológico, porém observamos que a combinação do diagnóstico citológico com análise da mutação melhorou significativamente todos os parâmetros analisados. A presença da mutação p.V600E em nossa casuística, não mostrou ser um fator isolado associado à pior prognóstico de CPT. Um maior número de pacientes e acompanhamento a longo prazo, somando-se cuidadosa avaliação clínica-morfológica com a detecção de mutação p.V600E e utilizando análises multivariadas, são necessários para esclarecer o significado prognóstico independente desta mutação. Estudos semelhantes também são necessários para encontrar uma maneira de combinar as características clínicas e ultrassonográficas, com a detecção da mutação p.V600E no material da PAAF para decidir a melhor abordagem cirúrgica / Thyroid cancer is the most common endocrine malignancy, accounting for 1- 2% of thyroid malignancies. Currently, the molecular pathogenesis of papillary thyroid carcinoma (PTC) has been linked to aberrant activation of the MAPK signaling pathway, triggered by mutations in several oncogenes. Of these, the p.V600E mutation of BRAF gene is the most frequent, being observed in 30%-80% of cases. Several studies have shown that the presence of this mutation is associated with an increased aggressiveness of the tumor and, consequently, a less favorable prognosis, making it an important set PTC. However, few methods used for analyzing samples from fine needle aspiration (FNA) of thyroid nodules were satisfactory regarding cost, time and sensitivity. The objectives of this study were to standardize the DNA extraction from samples obtained from ultrasound (US)-guided FNA of thyroid nodules; validate and determine the efficiency and cost-time of real-time PCR genotyping technique to detect p.V600E mutation from samples of FNA of thyroid nodules, to assess the prevalence of the mutation p.V600E mutation in patients with PTC, correlate the presence of the p.V600E mutation with clinical and histopathological features of higher aggressiveness and finally analyze the sensitivity, specificity and accuracy of cytological diagnosis in conjunction with molecular analysis of the p.V600E mutation in FNA material. Our series consisted of 224 patients, all underwent thyroidectomy, whose preoperative cytology was indeterminate (Bethesda classes III to V) or Bethesda VI (papillary carcinoma). We evaluated clinical data and hormone (TSH) and autoimmunity (anti-TPO and anti-TG), and the sonographic features (size, structure, echogenicity, presence of microcalcifications and halo and vasculature). The histological data were tumor size, histological variant, capsule invasion, lymphatic and vascular invasion, extrathyroidal extension, multicentricity and presence of malignant lymph nodes at surgery. Results: The patients were divided into benign (n:122) and malignant group (n:102), according to the final histological diagnosis. Malignant group had a mean age lower than the benign group (48.9 vs 54.2 years, p=0.008). There were no differences between groups regarding serum TSH (p=0.467), anti-TPO (p=0.535) and anti-TG (p=0.730). According to US characteristics, size and volume of the nodules were higher in the benign group (3.0 vs 2.6 cm and 12.38 cm 3 vs 14.5, p=0.008 and p<0.001, respectively). The nodules that showed hypoechogenicity, as well as the composition of solid nodules, the presence of microcalcifications, absence of hypoechoic halo and presence of central vascularization showed statistically higher frequency of malignancy. In the logistic regression model, older age, solid nodule without hypoechoic halo and microcalcifications were variables that influenced jointly in the presence of malignancy. In cytological diagnosis 78.6% (176/224) of nodules were evaluated as class III, IV and V, and of these 35.8% (63/176) had final histological diagnosis of malignant. The p.V600E mutation were identified in FNA material in 67.7% (69/102) of patients in malignant group, present in 70.3% (45/64) of papillary carcinoma classic variant and 69.7% (23/33) of follicular variant of papillary carcinoma, and all findings are confirmed in 100% of the samples through sequencing of the material obtained from the surgical tumor (fresh or paraffin). In patients with confirmed PTC (n:98), we compared patients with and without the mutation p.V600E according to clinical, histological poor prognosis, lymph node metastasis, and TNM stage according to AJCC. Only the presence of older age were significantly associated with the presence of the mutation p.V600E (p=0.041). We compared the cytological diagnosis based on the Bethesda classification and mutation analysis with the histological diagnosis p.V600E, the \"gold standard\" for diagnosis of PTC. Sensitivity, specificity, accuracy, positive and negative predictive value of cytological diagnosis was 67.4%, 94.4%, 79.8%, 93.3% and 71.2% respectively. Analysis of p.V600E mutation alone showed similar results to the cytological diagnosis, but we observed that the combination of cytological diagnosis with mutation analysis significantly improved all parameters analyzed. The presence of the mutation p.V600E in our series was not a single factor associated with worse prognosis of PTC. A larger number of patients and long term follow-up, adding to careful clinicalmorphological with p.V600E and mutation detection using multivariate analyzes are needed to clarify the independent prognostic significance of this mutation. Similar studies are also needed to find a combination among clinical and US, with p.V600E mutation detection in FNA material to decide the best surgical approach

This reseach firstly investigated iodine-induced apoptotic effects and the underlying mechanism in thyroid cancer cells. Results indicated that apoptosis induced by iodine, especially at high dose of iodine (100 muM), was mitochondrial-mediated, with the loss of mitochondrial membrane potential, Bak up-regulation, caspase 3 activation and cytochrome C release from mitochondria. Iodine treatment decreased the level of mutant p53 including the R273H mutant that possesses anti-apoptotic features while increased the p21 level. The block of p21 significantly prevented iodine-induced apoptosis. High doses of iodine also stimulated the transient activation of the subfamily members of MAPKs (ERK1/2, p38 and JNK1/2). The results showed the three subfamily members of MAPKs all worked as anti-apoptotic factors. Surprisingly, high doses of iodine promoted instead of suppressed the expression of anti-apoptotic protein Bcl-xL expression. The increase of Bc1-xL was likely to compensate the damage induced by iodine since the inhibition of Bc1-xL accelerated iodine-mediated apoptosis. Collectively, iodine induced mitochondrial-mediated apoptosis in thyroid cancer cells. This apoptotic pathway was involved in the activation of MAPKs pathways, which may subsequently up-regulate p21, Bc1-xL, and down-regulate anti-apoptotic mutant p53 expression. The findings provide solid molecular evidence to explain the epidemiological observation that iodine insufficiency promotes the thyroid tumor development. It may also reveal some novel molecular targets for the treatment of thyroid cancer. / Thyroid cancer is the most common endocrine malignancy and exhibits the full range of malignant behaviors from the relatively indolent occult differentiated thyroid cancer to uniformly aggressive and lethal anaplastic thyroid cancer. Iodine is a well known key element in thyroid normal function maintenance and thyroid cancer development. However, the mechanisms of iodine in thyroid cancer cells development are limited. Recent researches have indicated that iodine could induce cancer cells apoptosis, staying clear from the dysfunction of iodide-specific transportation systems in thyroid cancer cells. Thus, iodine-induced apoptosis may be an effective pathway for iodine to affect thyroid cancer development, but we know little about them. / To further explore iodine on the apoptotic effects of chemotherapeutic agents in thyroid cancer, anaplastic thyroid cancer cell line ARO was used. Anaplastic thyroid cancer is lethal because of its rapid progression and poor response to chemotherapy and radioiodine therapy. The study examined the effect of moderate dose of iodine (50 muM) on the apoptosis of ARO cells treated with doxorubicin (Dox) and histone deacetylase inhibitor sodium butyrate (NaB). The cytotoxic effect of either Dox or NaB alone was limited, but co-administration of NaB and Dox (NaB-Dox) significantly increased mitochondrial-mediated apoptosis. The effects of iodine to apoptosis-induced by the two agents were diversified. Iodine reduced the apoptosis induced by Dox or NaB-Dox but promoted apoptosis induced by NaB. To explain this diversifying finding, the experiment found that iodine exaggerated NaB-mediated Bcl-xL down-regulation. In contrast, it reduced the effect of Dox on the decrease of Bcl-xL expression. Further experiments showed that iodine regulated the level of Bcl-xL in ERK- or/and p38-related pathways. The balance between ERK and p38 may determine the iodine-modulated Bcl-xL expression. The high ERK/p38 activity ratio up-regulated Bc1-xL and enabled the tumor cells to resist chemotherapy, whereas the low ERK/p38 down-regulated Bc1-xL and sensitized the tumor cells to chemotherapy. Taken together, iodine plays a critical role in apoptosis of thyroid cancer cells induced by chemotherapeutic agents. The balance between ERK and p38 may determine cell survival and death through modulating Bcl-xL expression in thyroid cancer cells. The findings provide some new insights into the roles of iodine in chemotherapeutic agents-induced apoptosis in thyroid cancer cells. / To summarize, iodine-induced apoptotic effects on thyroid cancer cells is a key pathway for iodine to influence thyroid cancer development and chemotherapy. Meanwhile MAPKs-related mutant p53, p21 and Bcl-xL expression are critical in deciding thyroid cancer cells survival and death. Moreover, iodine can influence chemotherapeutic agents-induced apoptosis through ERK/p38-mediated Bcl-xL expression. / Liu, Xiaohong. / "December 2009." / Adviser: Charles Andrew van Hasselt. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O câncer da tireoide é a neoplasia endócrina mais comum, sendo responsável por cerca de 1 a 2% das neoplasias malignas da tireoide. Atualmente, a patogênese molecular do carcinoma papilífero da tireoide (CPT) tem sido relacionada à ativação aberrante da via de sinalização MAPK, desencadeada por mutações em diversos oncogenes. Destas, a mutação p.V600E do gene BRAF é a mais freqüente, sendo observada em 30%-80% dos casos. Numerosos estudos têm demonstrado que a presença dessa mutação está relacionada a uma maior agressividade do tumor e, conseqüentemente, a um prognóstico menos favorável, tornando-a um marcado importante no CPT. Contudo, poucos métodos utilizados na análise do gene BRAF em amostras de punção de nódulos tireoidianos foram satisfatórios em relação ao custo-tempo e sensibilidade do teste. Os objetivos deste estudo foram padronizar a extração de DNA a partir de amostras obtidas de PAAF guiada por ultrassom de nódulos tireoidianos; validar e determinar a eficiência e a relação custo-tempo da técnica de genotipagem por PCR em tempo real na detecção da mutação p.V600E do gene BRAF em amostras de PAAF de nódulos tireoidianos; analisar a prevalência da mutação p.V600E em pacientes com CPT; correlacionar à presença da mutação p.V600E com características clínicas e histopatológicas de maior agressividade e por fim analisar a sensibilidade, especificidade e acurácia do diagnóstico citológico em conjunto com a análise molecular da mutação p.V600E em material de PAAF. Nossa casuística foi composta por 224 pacientes, todos submetidos à tireoidectomia, cuja citologia pré-operatória foi indeterminada (Bethesda classes III a V) ou Bethesda VI (carcinoma papilífero). Foram avaliados dados clínicos e hormonais (TSH) e autoimunidade (anti-TPO e anti-TG), além das características ultrassonográficas (tamanho, estrutura, ecogenicidade, presença de microcalcificação e halo e vascularização). Os dados histológicos avaliados foram tamanho do tumor, variante histológica, invasão de cápsula, invasão vascular e linfática, extensão extratireoidiana, multicentricidade e presença de acometimento ganglionar à cirurgia. Os pacientes foram divididos em grupo benigno (n:122) e maligno (n:102), de acordo com o diagnóstico histológico final. O grupo maligno apresentou uma média de idade menor que o grupo benigno (48,9 vs 54,2 anos; p=0,008). Não observamos diferença entre os grupos com relação à dosagem sérica de TSH (p=0,467), anti-TPO (p=0,535) e anti-TG (p=0,730). Analisando as características ultrassonográficas, o tamanho e o volume dos nódulos foram maiores no grupo benigno (3,0 vs 2,6 cm e 12,38 vs 14,5 cm3; p=0,008 e p<0,001, respectivamente). Os nódulos que apresentaram hipoecogenicidade, assim como os nódulos de composição sólida, a presença de microcalcificações, ausência de halo hipoecogênico e presença de vascularização central apresentaram estatisticamente maior freqüência de malignidade. Em modelo de regressão logística, maior idade, nódulo sólido, sem halo hipoecogênico e com microcalcificações foram variáveis que influenciaram conjuntamente na presença de malignidade. No diagnóstico citológico 78,6% (176/224) dos nódulos avaliados foram classe III, IV e V, sendo que destes 35,8% (63/176) apresentaram diagnóstico histológico final maligno. Identificamos a mutação p.V600E no material de PAAF em 67,7% (69/102) dos pacientes do grupo maligno, estando presente em 70,3% (45/64) dos carcinomas papiliferos variante clássica e em 69,7% (23/33) dos carcinomas papilíferos variante folicular, sendo todos os achados confirmados em 100% das amostras através seqüenciamento automático do material obtido do tecido nodular a fresco ou parafinado dos pacientes. Nos pacientes com diagnóstico histológico de CPT (n:98), comparamos os pacientes com e sem a mutação p.V600E com relação aos dados clínicos, histológicos de pior prognóstico, presença de metástase linfonodal, classificação TNM e estádio de acordo com AJCC. Apenas a presença de idade mais avançada apresentou associação estatisticamente significativa com a presença da mutação p.V600E (p=0,041). Comparamos o diagnóstico citológico baseado na classificação de Bethesda e a análise da mutação p.V600E com o diagnóstico histológico considerado o padrão ouro para o diagnóstico de CPT. A sensibilidade, especificidade, acurácia, valor preditivo positivo e negativo do diagnóstico citológico foi de 67,4%, 94,4%, 79,8%, 93,3% e 71,2% respectivamente. Análise da mutação p.V600E isoladamente apresentou resultados similares ao do diagnóstico citológico, porém observamos que a combinação do diagnóstico citológico com análise da mutação melhorou significativamente todos os parâmetros analisados. A presença da mutação p.V600E em nossa casuística, não mostrou ser um fator isolado associado à pior prognóstico de CPT. Um maior número de pacientes e acompanhamento a longo prazo, somando-se cuidadosa avaliação clínica-morfológica com a detecção de mutação p.V600E e utilizando análises multivariadas, são necessários para esclarecer o significado prognóstico independente desta mutação. Estudos semelhantes também são necessários para encontrar uma maneira de combinar as características clínicas e ultrassonográficas, com a detecção da mutação p.V600E no material da PAAF para decidir a melhor abordagem cirúrgica / Thyroid cancer is the most common endocrine malignancy, accounting for 1- 2% of thyroid malignancies. Currently, the molecular pathogenesis of papillary thyroid carcinoma (PTC) has been linked to aberrant activation of the MAPK signaling pathway, triggered by mutations in several oncogenes. Of these, the p.V600E mutation of BRAF gene is the most frequent, being observed in 30%-80% of cases. Several studies have shown that the presence of this mutation is associated with an increased aggressiveness of the tumor and, consequently, a less favorable prognosis, making it an important set PTC. However, few methods used for analyzing samples from fine needle aspiration (FNA) of thyroid nodules were satisfactory regarding cost, time and sensitivity. The objectives of this study were to standardize the DNA extraction from samples obtained from ultrasound (US)-guided FNA of thyroid nodules; validate and determine the efficiency and cost-time of real-time PCR genotyping technique to detect p.V600E mutation from samples of FNA of thyroid nodules, to assess the prevalence of the mutation p.V600E mutation in patients with PTC, correlate the presence of the p.V600E mutation with clinical and histopathological features of higher aggressiveness and finally analyze the sensitivity, specificity and accuracy of cytological diagnosis in conjunction with molecular analysis of the p.V600E mutation in FNA material. Our series consisted of 224 patients, all underwent thyroidectomy, whose preoperative cytology was indeterminate (Bethesda classes III to V) or Bethesda VI (papillary carcinoma). We evaluated clinical data and hormone (TSH) and autoimmunity (anti-TPO and anti-TG), and the sonographic features (size, structure, echogenicity, presence of microcalcifications and halo and vasculature). The histological data were tumor size, histological variant, capsule invasion, lymphatic and vascular invasion, extrathyroidal extension, multicentricity and presence of malignant lymph nodes at surgery. Results: The patients were divided into benign (n:122) and malignant group (n:102), according to the final histological diagnosis. Malignant group had a mean age lower than the benign group (48.9 vs 54.2 years, p=0.008). There were no differences between groups regarding serum TSH (p=0.467), anti-TPO (p=0.535) and anti-TG (p=0.730). According to US characteristics, size and volume of the nodules were higher in the benign group (3.0 vs 2.6 cm and 12.38 cm 3 vs 14.5, p=0.008 and p<0.001, respectively). The nodules that showed hypoechogenicity, as well as the composition of solid nodules, the presence of microcalcifications, absence of hypoechoic halo and presence of central vascularization showed statistically higher frequency of malignancy. In the logistic regression model, older age, solid nodule without hypoechoic halo and microcalcifications were variables that influenced jointly in the presence of malignancy. In cytological diagnosis 78.6% (176/224) of nodules were evaluated as class III, IV and V, and of these 35.8% (63/176) had final histological diagnosis of malignant. The p.V600E mutation were identified in FNA material in 67.7% (69/102) of patients in malignant group, present in 70.3% (45/64) of papillary carcinoma classic variant and 69.7% (23/33) of follicular variant of papillary carcinoma, and all findings are confirmed in 100% of the samples through sequencing of the material obtained from the surgical tumor (fresh or paraffin). In patients with confirmed PTC (n:98), we compared patients with and without the mutation p.V600E according to clinical, histological poor prognosis, lymph node metastasis, and TNM stage according to AJCC. Only the presence of older age were significantly associated with the presence of the mutation p.V600E (p=0.041). We compared the cytological diagnosis based on the Bethesda classification and mutation analysis with the histological diagnosis p.V600E, the \"gold standard\" for diagnosis of PTC. Sensitivity, specificity, accuracy, positive and negative predictive value of cytological diagnosis was 67.4%, 94.4%, 79.8%, 93.3% and 71.2% respectively. Analysis of p.V600E mutation alone showed similar results to the cytological diagnosis, but we observed that the combination of cytological diagnosis with mutation analysis significantly improved all parameters analyzed. The presence of the mutation p.V600E in our series was not a single factor associated with worse prognosis of PTC. A larger number of patients and long term follow-up, adding to careful clinicalmorphological with p.V600E and mutation detection using multivariate analyzes are needed to clarify the independent prognostic significance of this mutation. Similar studies are also needed to find a combination among clinical and US, with p.V600E mutation detection in FNA material to decide the best surgical approach

Source: Masters Abstracts International, Volume: 47-06, page: 3373. Adviser: Wenyaw Chan. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

O iodo é transportado para a célula tireoideana através do simportador sódio-iodo (NIS). Estudo anterior de nosso grupo mostrou que a expressão do RNAm de NIS estava reduzida nos tecidos tumorais (T) tireoideanos quando comparada à dos tecidos não tumorais (NT) adjacentes. A metilação de ilhas CpG localizadas em promotores é um dos mecanismos epigenéticos que regula a expressão gênica. Já foi demonstrado que a metilação aberrante desempenha papel importante na tumorigênese humana, porém, estudos qualitativos não conseguiram definir um padrão de metilação do promotor de NIS no câncer diferenciado de tireóide. Assim, o presente estudo visou (1) investigar o padrão de metilação do promotor do gene NIS em amostras T (benignos e malignos) e NT adjacentes, e correlacionar o grau de metilação com os valores de RNAm, e (2) identificar novas sequencias regulatórias, alvos de metilação do DNA, que pudessem também modular a expressão de NIS. Foram estudados 30 pares de amostras de tecido tireoideano T e NT adjacente, sendo 10 benignos e 20 malignos. Também foi avaliado um par de amostras de paciente com nódulo maligno com alta captação de iodo (hipercaptante). A metilação da ilha1 CpG foi avaliada por PCR Metilação Específica (MSP) semiquantitativa e a metilação da ilha2 CpG foi analisada por bissulfito seqüenciamento; ambas com DNA bissulfito convertido. Novas ferramentas de bioinformática foram utilizadas na análise in silico para identificar ilhas CpG funcionais na região 5\' upstream da seqüência promotora de NIS. Plasmídios foram construídos contendo o fragmento de DNA da Ilha2 CpG na frente de gene reporter (luciferase) e usados em transfecções de células HEK293 para avaliar a atividade regulatória desta ilha. Celulas tumorais com baixa expressão de NIS foram tratadas com agente desmetilante 5Aza e a expressão de RNAm de NIS foi avaliada por PCR em tempo real. Na ilha1 CpG, todas as amostras (T e NT) do grupo benigno e maligno apresentaram metilação, não observando-se diferença no grau de metilação entre os grupos. No entanto, maiores níveis de metilação foram detectados na região 5 desta ilha, que decresceram na direção da extremidade 3. Observou-se maior grau de metilação no tecido T em relação ao tecido NT adjacente (T>NT) em 60% das amostras do grupo benigno e em 35% do grupo maligno. Não foi identificada correlação entre o grau de metilação da ilha1 CpG e a expressão de RNAm de NIS. Nas amostras (T e NT) do paciente com tumor hipercaptante não foi detectada metilação nesta ilha. Assim foi realizada nova análise in silico, a qual identificou por primeira vez uma segunda ilha CpG, ilha2 CpG, com 256pb, localizada entre as posições -2152 e -1887 (em relação ao sítio ATG), contendo 14 sítios CpG. As analises da metilação da ilha2 CpG indicaram que essa região também é alvo de metilação, sendo observada hipermetilação (66.07%) em todas as amostras T quando comparada às amostras NT(23,21%), nos grupos benigno e maligno. A análise individual de cada par de amostras detectou um padrão de metilação do gene NIS em tecidos tireoideanos; sendo que 100% das amostras do grupo benigno e 95% das do grupo maligno apresentavam a ilha2 CpG mais metilada no tecido T do que no tecido NT (T>NT). Além disso, foi possível identificar correlação inversa significativa entre o grau de metilação da ilha2 CpG e a expressão gênica de NIS. Estudos in vitro para caracterização funcional da ilha CpG2 mostraram que esta nova ilha pode ser considerada como um enhancer do gene NIS na presença de fatores de transcrição da tireóide. Também foi possível detectar, em linhagem de carcinoma folicular da tireoide, a reexpressão do RNAm de NIS paralelamente à redução do grau de metilação da ilha2 CpG, após o tratamento com agente desmetilante. Sendo assim, confirmamos que a metilação do promotor NIS é um evento freqüente em amostras T (benignas e malignas) e NT. Este é o o primeiro trabalho que descreve a existência da ilha2 CpG, na região 5 do gene NIS, com atividade de enhancer, que regularia a expressão gênica por metilação do DNA. Pela primeira vez identificou-se a existência de um padrão de metilação no gene NIS em tecido tireoideanos (T>NT), assim como correlação inversa entre o grau de metilação da ilha2 CpG e a expressão de RNAm de NIS. Estes resultados poderiam explicar a reduzida expressão do NIS observada em amostras tumorais benignas e malignas e a baixa captação de iodo dos tumores tireoideanos / Iodide is transported from the blood into thyroid cell through the sodium iodide symporter (NIS). We have previously detected reduced NIS mRNA expression in thyroid tumoral tissue (T) when compared with the non tumoral tissue (NT). Methylation of DNAs CpG islands, very common in promoters, is an epigenetic alteration that regulates gene expression. Aberrant gene methylation plays an important role in human tumorigenesis. Previous qualitative studies have failed to define a NIS promoter methylation pattern in thyroid cancer. This study aimed (1) to investigate the methylation pattern of the NIS gene promoter in thyroid tumors (benign and malignant), and to correlate the methylation status with cellular levels of NIS mRNA, in T and adjacent NT samples; (2) to identify new regulatory DNA sequences that could modulate NIS gene expression by methylation of the DNA. Thirty pairs of thyroid samples (tumoral and non-tumoral), 10 benign and 20 malignant tumors were included. A pair of samples of a malignant nodule with high iodide uptake was also included. Methylation of CpG Island1 was evaluated by semiquantitative Methylation Specific PCR (MSP) and methylation of CpG island2 was analyzed by bisulfite sequencing; both using bisulfite converted DNA. New bioinformatic tools were used in in silico analysis to identify functional CpG islands in the 5 upstream region of the NIS promoter. Plasmids were constructed containing the CpG island2 DNA fragment in front of a reporter gene (lucyferase) and used in transfections assays of HEK293 cells to assess the regulatory activity of this island. Tumor thyroid cell cultures with low NIS expression were treated with the demethylating agent 5Aza and NIS mRNA expression was quantified by real time PCR. Methylation of CpG island1 was detected in all the samples (NT and T) of benign and malignant group, no differences were observed in the methylation degree between the groups. However, highest methylation levels were detected in the 5 region of CpG island1, which decreased to the 3 end. The analysis of each pair of samples revealed higher methylation degree in T tissue with respect to NT adjacent tissue (T> NT) in 60% of the benign group and in 35% of the malignant group. No quantitative correlation was found between CpG island1 methylation and NIS mRNA levels in both groups. Methylation was not detected in the NT and T samples of the patient with high iodine uptake. Furthermore, new in silico analysis allowed to identify a second CpG island, CpG island2, with 256pb, located between positions - 2152 and -1887 (relative to ATG), containing 14 CpG sites. The analysis of the CpG island2 indicated that this region is also a target of DNA methylation. Hypermethylation was observed in all T samples (66.07%) when compared with NT samples (23.21%), in benign and malignant groups. The analysis of each pair of samples allowed to detect a NIS gene methylation pattern in thyroid tissues. In 100% of the benign group and in 95% of the malignant group methylation degree of the CpG island2 was higher in the T tissue as compared to NT tissue (T>NT). Moreover, it was possible to identify significant negative correlation between the methylation degree of CpG island2 and NIS mRNA gene expression. In vitro functional characterization of the CpG island2 showed that this new island may be considered an enhancer of NIS gene in the presence of thyroid transcription factors. The treatment of follicular thyroid carcinoma cells with demethylating agent restored the NIS mRNA expression, in parallel with the reduction of the CpG island2 methylation degree. In conclusion, we confirm that the NIS promoter methylation is a frequent event in thyroid T (benign and malignant) and NT samples. This is the first description of a new second CpG island in the 5\'region of the NIS gene, with enhancer activity, which may regulate gene expression by DNA methylation. This is the first report that described the existence of a NIS gene methylation pattern of in thyroid tissue (T>NT) as well as a negative correlation between the methylation degree of CpG island2 and NIS mRNA expression. These results may explain the reduced NIS expression observed in benign and malignant tumor samples as well as the low iodine uptake of thyroid tumors