Dominant-activating mutations in the RET (rear-ranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, is often associated with the development of medullary thyroid carcinoma (MTC). The proximal promoter region of the RET gene consists of a guanine-rich sequence containing five runs of three consecutive guanine residues that serve as the binding site for transcriptional factors. As we have recently shown, this stretch of nucleotides in the promoter region is highly dynamic in nature and tend to form non-B DNA secondary structures called G-quadruplexes, which suppress the transcription of the RET gene. In the present study, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that the incorporation of a piperidine ring in an ellipticine derivative, NSC311153 improves its binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound also interfered with the transcriptional mechanism of the RET gene in an MTC derived cell line, TT cells and significantly decreased the endogenous RET protein expression. We demonstrated the specificity of NSC311153 by using papillary thyroid carcinoma (PTC) cells, the TPC1 cell line which lacks the G-quadruplex forming sequence in the promoter region due to chromosomal rearrangement. The RET downregulation selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In the present study, we also showed that the systemic administration of a water soluble NSC311153 analog in a mouse MTC xenograft model inhibited the tumor growth through RET downregulation.

Resumo: O carcinoma medular de tireóide familiar (CMTF) é uma doença autossômica dominante relacionada a mutações do proto-oncogene RET, que ocorre principalmente nas regiões cisteínas da proteína RET dependentes dos códons 10 e 11. Responde por 5 a 15% dos casos de CMT hereditários. Para caracterizar o CMT de natureza familiar, este deve estar presente em pelo menos 4 indivíduos de uma mesma família. O presente trabalho objetivou o estudo clínico-genético de uma família portadora de carcinoma medular de tireóide. Esta família consiste de 25 indivíduos (13 homens e 2 mulheres) distribuídos em três gerações. Destes, seis apresentam a doença ativa: um homem da 1ª geração e dois homens e três mulheres da 2ª geração. Nenhum membro da 3ª geração desenvolveu a doença até agora. Foram coletadas amostras de sangue periférico de 19 desses indivíduos. Nestas amostras foram realizadas amplificações dos segmentos gênicos de interesse pela técnica da reação em cadeia da polimerase (PCR) e os respectivos seqüenciamentos. Até o momento, detectou-se em dois indivíduos, mutação no códon 10 (cys611tyr). Esta mutação tem uma incidência baixa (1-3%) nos casos de CMTF, mas, no entanto, implica em alto risco de agressividade do tumor. Além disso, detectou-se a presença de 3 polimorfismos (G691S, L769L, S904S), nos éxons 11, 13 e 15. Enquanto que a mutação cys611tyr está relacionada ao desenvolvimento do CMTF, sua relação com os polimorfismos encontrados e o papel desta relação com esses tumores não está esclarecida. A realização do rastreamento genético das mutações no RET deve ser considerada pela sua praticidade, custo-benefício e sensibilidade e especificidade do método. Além disso, a determinação de mutações nos familiares com CMT implicará em indicação cirúrgica preventiva e aconselhamento genético familiar. / Abstract: Familial medullary thyroid carcinoma (FMTC) is an autosomic dominant disorder related to mutations in the RET proto-oncogene. These mutation occur mostly in the extracellular rich cistein domains of the RET protein, related to the codons 10 and 11. The FMTC represents 5-15% of hereditary medullary thyroid carcinoma (MTC). To characterize MTC of familial origin, at least four members of a family must be affected. The present study focused upon clinical-genetics characteristics of a 25-member family (13 men and 12 women) distributed through three generations. Six of them had MTC: one first generation man, two men and three women from the second generation. Up until now, no member from the third generation has developed the disease. We studied 19 of these individuals for mutations in the RET protooncogene. A cys611tyr mutation was found in two of the kindreds. This mutation has a low incidence (1-3%) in FMTC, but implies in high aggressiveness risk. Moreover, we found the polymorphisms G691S, L769L, S904S in the exons 11, 13 and 15, respectively. While cys611tyr mutation is responsible for cases of FMTC, its relationship to these polymorphisms and the consequences of this interrelation to tumor development is still unknown. The screening for RET proto-oncogene mutations in relatives of MTC patients must be considered for its practicability, low cost, high specificity and sensibility. Besides, the determination of mutations in FMTC patients' kindred will conduct them to prophylactic surgery and familial counseling. / Orientador: Célia Regina Nogueira / Coorientador: Denise Perone / Banca: Glaúcia Mozeto / Banca: Laura Ward / Doutor

Medullary thyroid carcinoma (MTC) is a rare malignant tumor originating from thyroid parafollicular C cells. This tumor accounts for 3-4% of thyroid gland neoplasias. MTC may occur sporadically or inherited. The hereditary MTC is part of syndromes of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC). Germline mutations of the RET (REarranged during Transfection) protooncogene cause hereditary form of cancer, whereas somatic mutations can be present in sporadic form of the disease. The RET gene encodes a receptor tyrosine kinase involved in the activation of intracellular signaling pathways leading to proliferation, growth, differentiation, migration and survival. Nowadays, the only possibility of cure for MTC patients consists of total thyroidectomy associated with lymph node dissection. Based on the knowledge of the pathogenic mechanisms of MTC, new drugs have been developed in attempt to control metastatic disease. Of these, the small-molecule tyrosine kinase inhibitors (TKIs) represent one of the most promising agents for MTC treatment and clinical trials have shown encouraging results. Hopefully, the cumulative knowledge about the targets of action of these drugs as well as TKI-associated side effects will help on choosing the best therapeutic approach in order to enhance its benefits.

Medullary thyroid carcinoma (MTC) is a rare malignant tumor originating from thyroid parafollicular C cells. This tumor accounts for 3-4% of thyroid gland neoplasias. MTC may occur sporadically or inherited. The hereditary MTC is part of syndromes of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC). Germline mutations of the RET (REarranged during Transfection) protooncogene cause hereditary form of cancer, whereas somatic mutations can be present in sporadic form of the disease. The RET gene encodes a receptor tyrosine kinase involved in the activation of intracellular signaling pathways leading to proliferation, growth, differentiation, migration and survival. Nowadays, the only possibility of cure for MTC patients consists of total thyroidectomy associated with lymph node dissection. Based on the knowledge of the pathogenic mechanisms of MTC, new drugs have been developed in attempt to control metastatic disease. Of these, the small-molecule tyrosine kinase inhibitors (TKIs) represent one of the most promising agents for MTC treatment and clinical trials have shown encouraging results. Hopefully, the cumulative knowledge about the targets of action of these drugs as well as TKI-associated side effects will help on choosing the best therapeutic approach in order to enhance its benefits.

Made available in DSpace on 2014-06-11T19:31:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-27Bitstream added on 2014-06-13T20:41:56Z : No. of bitstreams: 1 colenci_bpc_dr_botfm.pdf: 920335 bytes, checksum: 5efcb4f17d304fb708911364da3ccb52 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O carcinoma medular de tireóide familiar (CMTF) é uma doença autossômica dominante relacionada a mutações do proto-oncogene RET, que ocorre principalmente nas regiões cisteínas da proteína RET dependentes dos códons 10 e 11. Responde por 5 a 15% dos casos de CMT hereditários. Para caracterizar o CMT de natureza familiar, este deve estar presente em pelo menos 4 indivíduos de uma mesma família. O presente trabalho objetivou o estudo clínico-genético de uma família portadora de carcinoma medular de tireóide. Esta família consiste de 25 indivíduos (13 homens e 2 mulheres) distribuídos em três gerações. Destes, seis apresentam a doença ativa: um homem da 1ª geração e dois homens e três mulheres da 2ª geração. Nenhum membro da 3ª geração desenvolveu a doença até agora. Foram coletadas amostras de sangue periférico de 19 desses indivíduos. Nestas amostras foram realizadas amplificações dos segmentos gênicos de interesse pela técnica da reação em cadeia da polimerase (PCR) e os respectivos seqüenciamentos. Até o momento, detectou-se em dois indivíduos, mutação no códon 10 (cys611tyr). Esta mutação tem uma incidência baixa (1-3%) nos casos de CMTF, mas, no entanto, implica em alto risco de agressividade do tumor. Além disso, detectou-se a presença de 3 polimorfismos (G691S, L769L, S904S), nos éxons 11, 13 e 15. Enquanto que a mutação cys611tyr está relacionada ao desenvolvimento do CMTF, sua relação com os polimorfismos encontrados e o papel desta relação com esses tumores não está esclarecida. A realização do rastreamento genético das mutações no RET deve ser considerada pela sua praticidade, custo-benefício e sensibilidade e especificidade do método. Além disso, a determinação de mutações nos familiares com CMT implicará em indicação cirúrgica preventiva e aconselhamento genético familiar. / Familial medullary thyroid carcinoma (FMTC) is an autosomic dominant disorder related to mutations in the RET proto-oncogene. These mutation occur mostly in the extracellular rich cistein domains of the RET protein, related to the codons 10 and 11. The FMTC represents 5-15% of hereditary medullary thyroid carcinoma (MTC). To characterize MTC of familial origin, at least four members of a family must be affected. The present study focused upon clinical-genetics characteristics of a 25-member family (13 men and 12 women) distributed through three generations. Six of them had MTC: one first generation man, two men and three women from the second generation. Up until now, no member from the third generation has developed the disease. We studied 19 of these individuals for mutations in the RET protooncogene. A cys611tyr mutation was found in two of the kindreds. This mutation has a low incidence (1-3%) in FMTC, but implies in high aggressiveness risk. Moreover, we found the polymorphisms G691S, L769L, S904S in the exons 11, 13 and 15, respectively. While cys611tyr mutation is responsible for cases of FMTC, its relationship to these polymorphisms and the consequences of this interrelation to tumor development is still unknown. The screening for RET proto-oncogene mutations in relatives of MTC patients must be considered for its practicability, low cost, high specificity and sensibility. Besides, the determination of mutations in FMTC patients’ kindred will conduct them to prophylactic surgery and familial counseling.

Medullary thyroid carcinoma (MTC) is a rare malignant tumor originating from thyroid parafollicular C cells. This tumor accounts for 3-4% of thyroid gland neoplasias. MTC may occur sporadically or inherited. The hereditary MTC is part of syndromes of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC). Germline mutations of the RET (REarranged during Transfection) protooncogene cause hereditary form of cancer, whereas somatic mutations can be present in sporadic form of the disease. The RET gene encodes a receptor tyrosine kinase involved in the activation of intracellular signaling pathways leading to proliferation, growth, differentiation, migration and survival. Nowadays, the only possibility of cure for MTC patients consists of total thyroidectomy associated with lymph node dissection. Based on the knowledge of the pathogenic mechanisms of MTC, new drugs have been developed in attempt to control metastatic disease. Of these, the small-molecule tyrosine kinase inhibitors (TKIs) represent one of the most promising agents for MTC treatment and clinical trials have shown encouraging results. Hopefully, the cumulative knowledge about the targets of action of these drugs as well as TKI-associated side effects will help on choosing the best therapeutic approach in order to enhance its benefits.

Genetic causes of medullary thyroid carcinoma and Hirschsprung's disease Abstract Medullary thyroid carcinoma (MTC) and Hirschsprung's disease (HSCR) are classified as simple neurocristopathies, i.e. diseases linked to neural crest-derived cells. MTC is derived from parafollicular cells of the thyroid and HSCR is characterized by absence of enteric ganglia in the gastrointestinal tract. The RET proto-oncogene is only expressed in neural crest-derived cells, including parafollicular cells and enteric neurons. The RET encodes a transmembrane tyrosinekinase receptor that plays an important role during proliferation, differentiation and cell survival, and activates many signaling pathways. If the strictly regulated activation fails, e.g. due to mutations in the specific gene locations, the RET becomes a highly effective oncogene. Activating germline mutations in the RET proto- oncogene lead to hereditary forms of MTC, whereas sporadic forms of MTC are caused by somatic mutations in the tumor tissue. On the contrary, inactivating mutations induce migration failure of ganglion cell precursors during the development of enteric nervous system and result in the development of HSCR. In rare cases, the coexistence of both diseases is caused by mutations with a dual gain-of-function and loss-of-function character....

Small cell variant of medullary thyroid carcinoma is an extremely rare histologic entity with a paucity of data. As such, there is a lack of literature and clinical experience regarding this disease. In this report, we examine a case of small cell variant of medullary thyroid carcinoma that presented with intractable nausea, vomiting and diarrhea. While these symptoms were essentially refractory to the standard symptomatic treatment, further laboratory analysis revealed dramatically elevated calcitonin levels and mildly raised thyroid-stimulating hormone levels. Interestingly, repletion of thyroid hormone and treatment with lanreotide resulted in an abatement of our patient's symptoms. This temporal clinical improvement highly suggests a potential role involving thyroid-stimulating hormone and calcitonin levels in the pathogenesis of this disease, and consequently suggests a role for thyroxine in treating the associated gastrointestinal symptoms.

Le cancer médullaire de la thyroïde (CMT), qui représente 5 à 8% des cancers de la thyroïde, est issu de la transformation maligne des cellules C du parenchyme thyroïdien. Ce cancer, sporadique dans 70 à 80% des cas et familial pour les 20 à 30% restants, est essentiellement lié à des anomalies du proto-oncogène RET, codant un récepteur à activité tyrosine kinase. La fréquence élevée des mutations activatrices de RET ont permis d’identifier ce récepteur comme une cible thérapeutique majeure. Si la chirurgie est le traitement de référence pour les formes localisées, les formes localement avancées ou métastatiques, de pronostic plus péjoratif étaient avant le développement des thérapies moléculaires ciblées, dans une impasse thérapeutique. La meilleure compréhension de la biologie des tumeurs a permis le développement de ces thérapies plus rationnelles et plus spécifiques, en particulier des inhibiteurs d’activité tyrosine kinase (ITK). L’optimisation de leur utilisation en clinique nécessite de mieux comprendre leurs mécanismes d’action.Dans ce contexte, les objectifs de cette thèse ont été à la fois cognitifs et cliniques, visant à améliorer la compréhension de la réponse moléculaire à deux ITKs, le sunitinib et la vandetanib,dans le CMT, et à identifier de nouveaux biomarqueurs de suivi thérapeutique. Dans un premier temps, nous avons montré les effets antiprolifératifs, antitumoraux et antiangiogéniques du sunitinib et du vandetanib dans un modèle de CMT muté RETC634W, mettant en évidence des profils d’activité proches entre les deux inhibiteurs. Puis, les principales voies de signalisation mises en jeu lors de la réponse à ces ITKs ont été explorées par Reverse-Phase Protein Array(RPPA). Par une approche transcriptomique haut-débit menée sur des modèles précliniques, les principales fonctions cellulaires impliquées dans la réponse au sunitinib et au vandetanib ont été identifiées. Le rôle de gènes participant à l’invasion tissulaire et au pouvoir métastatique a été mis en évidence. De nouveaux biomarqueurs potentiels de réponse au vandetanib et au sunitinib, tels que l’IL-8 et le TGF-2 dont les taux sériques sont significativement plus élevés chez les patients atteints de CMT, ont été identifiés. Enfin, l’intérêt de trois approches méthodologiques dans lesuivi de la réponse antitumorale chez les patients a été évalué. Ainsi, le développement d’une méthode de dosage du vandetanib par spectrométrie de masse a permis de suggérer un lien entre des taux sériques élevés et l’apparition de toxicités sévères. L’évaluation de biomarqueurs dans le sérum de patients traités par le vandetanib a souligné l’intérêt de l’IL-8 comme marqueur pronostic potentiel dans cette pathologie. Enfin les résultats préliminaires, évaluant la réponse au sunitinib par échographie doppler sur un modèle préclinique de souris xénogreffées, ont confirmé l’intérêt de l’imagerie fonctionnelle dans ce domaine. / Medullary thyroid carcinoma (MTC) accounts for 5-8% of all thyroid cancers and occurs as either a sporadic form or in a familial context (25% of cases). Mutations which activate the RET proto-oncogene, encoding a tyrosine kinase receptor, are responsible for familial forms and are also detected in one-third of sporadic tumors. MTC patients with local disease may be cured after initial surgery, but persistent or recurrent disease occurs in half of cases, and distant metastases are the major cause of tumor related death. Up to now there is no effective systemic treatment and new therapeutic strategies are needed for locally advanced or metastatic MTC patients. The constitutive activation of RET is crucial in MTC pathogenesis and led to the development of small compounds targeting its tyrosine kinase activity (TKI). Gaining an understanding of how cancer cells respond to drugs is challenging to improve clinical use of these new therapeutic agents. In this context, we aimed to characterize molecular mechanisms of action of sunitinib and vandetanib, two TKIs currently evaluated in MTC patients. Our results, in in vitro as well as in vivo MTC models based on the RETC634W TT cell line, demonstrate that sunitinib and vandetanib has similar antiproliferative, antitumoral and antiangiogenic properties. Using the Reverse Phase Protein Array (RPPA) large-scale technology, we identified major signalling pathways inhibited after TKIs’ treatment. Expression microarrays allowed us to investigate signaling pathways modified after sunitinib and vandetanib treatment and to show that TKIs’ treatment induced major changes in the expression of genes involved in tissue invasion and metastasis. We identified encoding secreted proteins as candidate soluble biomarkers of response and, among them, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-2. Three modalities for determining early responses to targeted agents in MTC patients were evaluated. First, a sensitive mass spectrometry assay was developed for the quantitation of vandetanib, and applied in MTC patients, showing a potential relationship between toxic side-effects and vandetanib serum levels and suggesting that therapeutic drug monitoring may be a useful tool for MTC patients’ follow-up. Then, candidate biomarkers were investigated in MTC patients underlying the potential use of IL-8 as prognostic marker. Finally, using doppler imaging in xenografted mice model, we confirmed the utility of imaging techniques in clinical evaluation of TKI’s response.

Le carcinome médullaire de la thyroïde (CMT) est une tumeur neuroendocrine rare, se développant à partir des cellules sécrétant la calcitonine. Cette tumeur survient dans un contexte familial dans un tiers des cas. Toutes les formes germinales et près de 40% des formes sporadiques sont causées par une mutation ponctuelle activatrice de l’oncogène RET, codant pour un récepteur membranaire à activité tyrosine kinase. Les événements oncogéniques à l’origine des formes sporadiques non mutées RET restent mal définis, à l’exception de mutations activatrices des oncogènes RAS découvertes récemment.Le pronostic péjoratif du CMT est essentiellement lié à un envahissement ganglionnaire précoce. A ce titre, la chirurgie initiale est souvent insuffisante et les formes métastatiques ont longtemps été considérées en impasse thérapeutique. L’avènement récent des inhibiteurs séléctifs de tyrosine kinases (ITK) a apporté un nouvel élan à la prise en charge des tumeurs réfractaires, certains d’entre eux incluant dans leur spectre d’action le récepteur RET. Mais l’optimisation de ces traitements requiert une connaissance préalable des mécanismes moléculaires sous-jacents au développement tumoral.Dans ce contexte et en nous appuyant sur une collection importante de prélèvements humains, nous avons cherché à approfondir la decription du ‘paysage génomique’ du CMT.Dans un premier temps, nous avons évalué les anomalies structurales ponctuelles et chromosomiques présentées par les CMT. Nous avons montré, par optimisation de méthodes de séquençage, que les mutations des gènes RET et RAS interviennent dans plus de 96% des cas et que ces évènements sont mutuellement exclusifs. Ces mutations permettent de distinguer plusieurs groupes d’agressivité et de réponse aux traitements par ITK. Nous avons également observé - par technique d’hybridation génomique comparative - des anomalies de grande ampleur récurrentes dans cette pathologie : les délétions du bras court du chromosome 1 et des chromosomes entiers 4 et 22 apparaissent comme étant des évènements précoces et indépendants de la tumorigenèse du CMT.Dans un second temps, nous avons déterminé - par approche de type biopuce - les profils d’expression de microARN dans les CMT. Certains de ces régulateurs post-transcriptionnels majeurs semblent liés au caractère invasif de la tumeur, et notamment les miR-21, miR-199 et miR-129. Nous avons également démontré le potentiel d’utilisation des microARN miR-21 et miR-199 en tant que biomarqueurs circulants du CMT. L’impact fonctionnel des formes précurseurs mir-21 et mir-129 a ensuite été évalué par transfection dans les modèles cellulaires TT et MZ-CRC1.Les observations ainsi obtenues offrent de nombreuses perspectives d’études. Elles permettent la définition de marqueurs tissulaires distinguant a priori les tumeurs métastatiques et/ou réfractaires aux thérapies. Enfin, elles mettent en lumière de nouvelles pistes pour la découverte de cibles thérapeutiques additionnelles dans cette pathologie. / Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor, arising from calcitonin-secreting cells. This cancer occurs in a family context in a third of cases. All inherited forms and nearly 40% of sporadic forms are caused by activating point-mutations in the RET oncogene, coding for a tyrosine-kinase receptor. Other oncogenic events causing sporadic cases remain unclear, but activating mutations of RAS oncogenes have been discovered recently.Prognosis of MTC is essentially linked to early lymph node occurrence. Initial surgery of metastatic forms is often insufficient and patients are considered in therapeutic dead-end. The recent advent of selective tyrosine-kinase inhibitors (TKIs) has brought a new impetus to the management of refractory tumors, some of them targeting the RET receptor. Optimization of these treatments require improving knowledge of the underlying molecular mechanisms of tumor development.In this context and helped by a large collection of human specimens, we have sought to deepen the description of genomic landscape of MTC.At first, we evaluated the structural and chromosomal abnormalities presented by MTC. We showed, by optimizing sequencing methods, that RET and RAS mutations are involved in over 96% of the cases, these events are mutually exclusives. These mutations can distinguish several groups of aggressiveness and of response to TKI treatments. We also observed, by comparative genomic hybridization techniques, recurrent abnormalities such as deletion of the short arm of chromosome 1 and loss of entire chromosomes 4 and 22. These losses appear to be early events of tumorigenesis MTC.In a second step, we determined - by a microarray approach – the microRNA expression profile of MTC. Some of these post-transcriptional regulators seem related to tumor invasiveness, such as miR-21, miR-199 and miR-129. We demonstrated the potential of microRNAs miR-21 and miR-199 as circulating diagnosis biomarkers of MTC. The functional impact of the precursor forms mir-21 and mir-129 was then evaluated by transfection in TT and MZ- CRC1 cellular models.Observations obtained pave the way for a lot of new potential studies. They allow the definition of tissue biomarkers distinguishing metastatic forms or refractory patients. Finally, they highlight new pathways for the discovery of additional therapeutic targets in this disease.