Diagnostic, Prognostic and Therapeutic Value of Gene Signatures

Gene expression studies have revealed diagnostic profiles and upregulation of specific pathways in many solid tumors. Some gene-expression signatures are already used as predictors of relapse in early breast cancer patients. The explosion of new information in gene expression profiling could potentially lead to the development of tailored treatments in many solid tumors. In addition, many studies are ongoing to validate these signatures also in predicting response to hormonal, chemotherapeutic, and targeted agents in breast cancer as well as in other tumors. This book has been carried out with the aim of providing readers a useful and comprehensive resource about the range of applications of microarray technology on oncological diseases. The book is principally addressed to resident and fellow physicians, medical oncologists, molecular biologists, biotechnologists, and those who study oncological diseases. The chapters have been written by leading international researchers on these topics who have prepared their manuscripts according to current literature and field experience with microarray technology

Emerging roles of Keap1/Nrf2 signaling in the thyroid gland and perspectives for bench-to-bedside translation.

The signaling pathway centered on the transcription factor nuclear erythroid factor 2-like 2 (Nrf2) has emerged during the last 15 years as a target for the prevention and treatment of diseases broadly related with oxidative stress such as cancer, neurodegenerative and metabolic diseases. The roles of Nrf2 are expanding beyond general cytoprotection, and they encompass its crosstalk with other pathways as well as tissue-specific functions. The thyroid gland relies on reactive oxygen species for its main physiological function, the synthesis and secretion of thyroid hormones. A few years ago, Nrf2 was characterized as a central regulator of the antioxidant response in the thyroid, as well as of the transcription and processing of thyroglobulin, the major thyroidal protein that serves as the substrate for thyroid hormone synthesis. Herein, we summarize the current knowledge about the roles of Nrf2 in thyroid physiology, pathophysiology and disease. We focus specifically on the most recent publications in the field, and we discuss the implications for the preclinical and clinical use of Nrf2 modulators

Hyperthyroidism and Papillary Thyroid Carcinoma in Thyrotropin Receptor D633H Mutant Mice

Background: Constitutively active thyrotropin receptor (TSHR) mutations are the most common etiology of non-autoimmune hyperthyroidism (NAH). Thus far, the functionality of these mutations has been tested in vitro, but the in vivo models are lacking. Methods: To understand the pathophysiology of NAH, we introduced the patient-derived constitutively active TSHR D633H mutation into the murine Tshr by homologous recombination. Results: In this model, we observed both subclinical and overt hyperthyroidism depending on the age, sex and copy number of the mutated allele. Homozygous mice presented hyperthyroidism at 2 months of age, while heterozygous animals showed only suppressed TSH. Interestingly, at 6 months of age, thyroid hormone concentrations in all mutant mice were analogous to wildtypes, and they showed colloid goiter with flattened thyrocytes. Strikingly, at one-year of age nearly all homozygous mice presented large papillary thyroid carcinomas (PTC). Mechanistically, this PTC phenotype was associated with an overactive thyroid and strongly increased stainings of proliferation, pERK, and NKX2-1 markers, but no mutations in the “hot-spot” areas of common oncogenes (Braf, Nras, Kras) were found. Conclusions: this is the first study to reveal the dynamic age-, sex- and gene dosage-dependent development of NAH. Furthermore, we show that a constitutively active TSHR can trigger a malignant transformation of thyrocytes

Identification of diagnostic and predictive morphological and molecular markers in renal tumors

Objectives: Ongoing advances in tumor biology keep expanding the spectrum of renal neoplasms, with more than twenty renal cell carcinoma histotypes recognized in the last WHO classification. Thus, the discovery of novel diagnostic and predictive biomarkers for rarer tumors, such as those belonging to the “molecularly defined category”, is warranted to improve physicians’ ability to recognize and treat these uncommon diseases. Furthermore, even in the most common entities, a noteworthy percentage of the patients treated for an organ-confined or locally advanced neoplasm lately develop distant metastases. In the present project, we aimed to identify novel pathological, immunohistochemical, and molecular features bearing a diagnostic and predictive potential in a broad spectrum of common and rare epithelial or mesenchymal renal neoplasms. Material and methods: The following cases were retrieved, including samples from both primary neoplasms and metastases when available: four high-grade eosinophilic renal cell tumors, twenty-seven TFE3-rearranged renal cell carcinomas, ten TFEB-rearranged renal cell carcinomas, one TFE3-rearranged PEComa, thirteen FH-deficient renal cell carcinomas, and one hundred forty-six clear cell renal cell carcinomas. The specimens were extensively studied by immunohistochemistry (IHC), FISH, and their molecular analyses, including diagnostic and predictive markers, such as STING and PD-L1, and targetable molecular alterations, like mTOR mutations. Results: Concerning eosinophilic tumors, the cases herein described belong to a novel entity, provisionally named mTOR-mutated eosinophilic renal cell carcinoma, sharing metastatic potential, mTOR mutations, loss of chromosome 1, cathepsin-K expression, and response to target therapy. Cathepsin K, CA9, CK7, and parvalbumin are the most reliable markers in distinguishing TFE3/TFEB-rearranged renal cell carcinomas from common renal cell tumors; the positivity threshold depends on the marker considered. Renal TFE3-rearranged PEComas are usually composed of nested clear to eosinophilic cells and harbor SFPQ/PSF::TFE3 fusions in more than half of the cases. By IHC, they express cathepsin K, HMB45, MART-1, and CD68 (PG-M1) while labeled negative for PAX8, CD10, and CKs. Conversely, TFE3-rearranged renal cell carcinomas are positive for PAX8, CD10, and, less frequently, cathepsin K, HMB45, and MART-1, whereas they commonly stain negative for CD68 (PG-M1) and CKs. About FH-deficient renal cell carcinoma, 75% of our cases (8/12) developed metastases, mainly involving the abdominal lymph nodes (5/8, 67%), and the peritoneum (4/8, 50%). Metastases revealed morphological heterogeneous architecture often differing from the primary renal tumor, closely resembling a primary mesothelial process when occurring in the peritoneum. In all primary and secondary samples, the IHC combination of FH loss and positive 2SC staining was diagnostically helpful. STING staining was detected in most primary tumors (9/11, 82%), including all but two aggressive renal neoplasms and 86% (6/7) of tumors significantly expressing PD-L1. Finally, in clear cell renal cell carcinoma, STING immunolabeling was detected in 36% (53/146) of the samples, more frequently in high-grade (G3-G4) tumors (48%, 43/90) and recurrent/metastatic ones (75%, 24/32). Moreover, STING correlated with aggressiveness parameters, like granular necrosis (p = 0.001), stage (p < 0.001), and metastases (p < 0.001), and reached an independent statistical significance (p = 0.029) in multivariate analysis. Conclusions: The identification of reliable morphological, immunohistochemical, and molecular findings in kidney cancer is crucial to address the needs raised by evolving classifications. The diagnostic and predictive markers described in the present project will provide novel insight to improve the global management of kidney cancer patients, filling the gap between basic research and daily practice

The Keap1/Nrf2 Signaling Pathway in the Thyroid-2020 Update.

The thyroid gland has a special relationship with oxidative stress. On the one hand, like all other tissues, it must defend itself against reactive oxygen species (ROS). On the other hand, unlike most other tissues, it must also produce reactive oxygen species in order to synthesize its hormones that contribute to the homeostasis of other tissues. The thyroid must therefore also rely on antioxidant defense systems to maintain its own homeostasis in the face of continuous self-exposure to ROS. One of the main endogenous antioxidant systems is the pathway centered on the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1). Over the last few years, multiple links have emerged between the Keap1/Nrf2 pathway and thyroid physiology, as well as various thyroid pathologies, including autoimmunity, goiter, hypothyroidism, hyperthyroidism, and cancer. In the present mini-review, we summarize recent studies shedding new light into the roles of Keap1/Nrf2 signaling in the thyroid

The role of osteopontin in tumour progression and metastasis in breast cancer

The use of cancer biomarkers to anticipate the outlines of disease has been an emerging issue, especially as cancer treatment has made such positive steps in the last few years. Progress in the development of consistent malignancy markers is imminent because advances in genomics and bioinformatics have allowed the examination of immense amounts of data. Osteopontin is a phosphorylated glycoprotein secreted by activated macrophages, leukocytes, and activated T lymphocytes, and is present in extracellular fluids, at sites of inflammation, and in the extracellular matrix of mineralized tissues. Several physiologic roles have been attributed to osteopontin, i.e., in inflammation and immune function, in mineralized tissues, in vascular tissue, and in kidney. Osteopontin interacts with a variety of cell surface receptors, including several integrins and CD44. Binding of osteopontin to these cell surface receptors stimulates cell adhesion, migration, and specific signaling functions. Overexpression of osteopontin has been found in a variety of cancers, including breast cancer, lung cancer, colorectal cancer, stomach cancer, ovarian cancer, and melanoma. Moreover, osteopontin is present in elevated levels in the blood and plasma of some patients with metastatic cancers. Therefore, suppression of the action of osteopontin may confer significant therapeutic activity, and several strategies for bringing about this suppression have been identified. This review looks at the recent advances in understanding the possible mechanisms by which osteopontin may contribute functionally to malignancy, particularly in breast cancer. Furthermore, the measurement of osteopontin in the blood or tumors of patients with cancer, as a way of providing valuable prognostic information, will be discussed based on emerging clinical data.Fundação para a Ciência e a Tecnologia (FC

Molecular Alterations and Expression Dynamics in the Etiopathogenesis of Thyroid Cancer

Thyroid carcinoma is the most prevalent endocrine malignancy and accounts for 2% of all human cancers. In the past decade, knowledge of genetic alterations of thyroid cancer (TC) has rapidly expanded, which has provided new insights into thyroid cancer etiology and has offered novel diagnostic tools and prognostic markers that enable improved and personalized management of thyroid cancer patients. Alterations in key signaling effectors seem to be the hallmark of distinct forms of thyroid neoplasia. Mutations or rearrangements in genes that encode Mitogen activated protein kinase (MAPK) pathway effectors seem to be required for transformation. Mutations in BRAF were the most recently identified MAPK effector in thyroid cancer. BRAF V600E is the most common alteration in sporadic papillary carcinoma. Three RAS proto-oncogenes (NRAS, HRAS & KRAS) are implicated in human thyroid tumorigenesis. High incidence of thyroid cancer worldwide indicates the importance of studying genetic alterations that lead to its carcinogenesis. BRAF and RAS alterations represent a novel indicator of the progression and aggressiveness of thyroid carcinogenesis. The GSα-adenylyl cyclase-cyclic AMP (cAMP) cascade is effected in thyroid cancer. Promoter hypermethylation of multiple genes especially TSHR has been identified to play a role in thyroid cancers, in particular showing a close association with BRAF mutational status. So, the main aim of the study was to elucidate the involvement of BRAF and RAS gene mutations along with BRAF expression and thyroid-stimulating hormone receptor (TSHR) hypermethylation in North Indian patients and investigate their association with clinicopathological characteristics

MiR-218 as a multifunctional regulator of oncogenic processes in different solid tumors

MicroRNAs are highly conserved small non-coding regulatory RNAs that involve in post transcriptional regulating of gene expression during different cellular mechanisms. Aberration of miR-218 expression during tumorigenesis of different solid tumors has been reported by numerous studies. In current systematic review article, by using the terms “miR-218” and “cancer” we first searched for English language articles in the PubMed database, published from 1993 to April 2014. Then by a comprehensive review of related articles, we provided some new insights that highlight novel features and functions of miR-218 in initiation and progression of solid tumors. The majority of these studies propose a tumor suppressing role for miR-218 considering the fact that it is significantly down-regulated in tumor tissues compared with normal specimens. Despite accumulating body of evidence regarding tumor suppressor functions of miR-218 in solid tumors; more intensive reviewing about available miR-218 recent original studies and interpretation of existing data, revealed the multifunctional role of miR-218 in these kinds of malignancies by targeting different corresponding target genes. Take all together, MiR-218 targets different cellular processes in cancer cells and its expression pattern is in an important association with various states and features of tumors. It seems that miR-218 can increase the speed of cell cycle and cell division in lower sample grades and along with progression of cancer cells it's function changes to stabilization the cancer cells and not allowing them to invade. thats why it often shows up-regulation in lower grades and down-regulation in metastatic phase. Therefore, it seems of great importance to check samples stage, grade, lymph node metastasis status and other tumor features before evaluation of miR-218 as a prognostic or diagnostic biomarker. © 2016, Iranian Neurogenetics Society. All rights reserved

Molecular characterisation of the poorly differentiated and undifferentiated thyroid carcinomas using genome-wide approaches

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2013Poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) are highly malignant tumours composed by dedifferentiated cells, for which current therapeutic options have been ineffective. In the present project, the molecular signatures and genetic alterations associated with these tumours were elucidated, by using genome-wide expression analysis as first assessment. The role of the microRNA stability in thyroid tumourigenesis was also evaluated. The comparison of the expression profiles between PDTC, well-differentiated thyroid carcinomas and normal thyroid tissues, and between ATC and normal thyroid tissues, revealed that PDTC and ATC have common deregulated signatures, indicative of cell adhesion impairment and increased cell cycle, proliferation and chromosomal instability. Additionally, ATC were specifically characterized by loss of epithelial and thyroid-related functions and activation of components from TGF-β pathway. The gene expression data suggested that follicular variants of papillary carcinomas were possible precursors of PDTC, whereas, ATC were more similar to classical papillary carcinomas. Validation by quantitative RT-PCR in independent sample sets, allowed to confirm the significant over-expression of UHRF1 (associated to proliferation) and downregulation of ITIH5 (associated to cell adhesion and invasion) in PDTC relatively to normal tissues. Moreover, CDKN3 gene, which encodes a cyclin-dependent kinase inhibitor, was shown to be significantly upregulated in PDTC and ATC relatively to normal thyroid, and in accordance with its suppressive function, CDKN3 was aberrantly spliced in ATC samples. The over-expression of the epithelial-tomesenchymal transition factor SNAI2, which is induced by TGF-β, was significant in ATC. Attending to the deregulated pathways identified, a mutational screening was undertaken in 26 ATC and 22 PDTC, which included the hot-spot regions of RAS, BRAF, TP53, CTNNB1 (β-catenin) and PIK3CA genes, and, for the first time, a comprehensive mutational analysis of cell cycle [CDKN1A (p21CIP1); CDKN1B (p27KIP1); CDKN2A (p14ARF, p16INK4A); CDKN2B (p15INK4B); CDKN2C (p18INK4C)], and cell adhesion regulators (AXIN1). Most mutations were identified in TP53 (42% of ATC; 27% of PDTC) and in RAS (31% of ATC; 18% of PDTC). The alterations in these genes were mutually exclusive (P=0.0354), and were associated with a lower patient survival (P=0.0383). CDKN2A and CDKN2B were found to be mutated, for the first time, in up to 25% of PDTC. Other known recurrent mutations in ATC (in BRAF, PIK3CA, CTNNB1 and AXIN1 genes) were rarely detected, undermining their role in ATC development. The study of the microRNA stability in thyroid tumourigenesis is still ongoing. Turnover rates were determined in a dedifferentiated thyroid carcinoma cell line (BCPAP), under standard conditions. A non-disruptive pulse-labelling method was applied and the decrease in microRNA expression along the time was quantified by two-colour arrays. These results represent the first report for an atlas of human microRNA half-lives. The determined average half-life for 249 microRNA was 2.5 days, a stability time more comparable to proteins than to mRNA. The comparison of these data with decay rates in normal thyroid cells will probably uncover miRNA for which stability is deregulated. In conclusion, this project presents several genes and molecular events that were found deregulated in PDTC and ATC. More importantly, these represent potential therapeutic targets that can be used for the treatment of these highly aggressive tumours, in the near future.O cancro da tiróide constitui a neoplasia mais comum do sistema endócrino (van der Zwan et al., 2012), representando nos homens e nas mulheres respectivamente, cerca de 1 e 3% dos casos de cancro, estimados em Portugal (Ferlay et al., 2010a) e no mundo (Ferlay et al., 2010b). Os tumores com origem no epitélio folicular representam 90 a 95% das neoplasias da tiróide (Nikiforov and Nikiforova, 2011), e de acordo com as características morfológicas e clínicas, os tumores foliculares malignos são subdivididos em carcinomas bem-diferenciados (WDTC), carcinomas pouco diferenciados (PDTC) e carcinomas indiferenciados ou anaplásicos (ATC). Os tumores foliculares da tiróide representam assim, um modelo clássico do processo tumoral pelo qual uma célula epitelial possui potencial para originar diferentes tipos de tumores, cada um com características patológicas distintas. Nos WDTC incluem-se os carcinomas papilares (PTC) e os carcinomas foliculares (FTC), sendo os PTC os mais frequentes, representando cerca de 80 a 90% dos casos (Kondo et al., 2006; Nikiforov and Nikiforova, 2011). Os WDTC são, em geral, tratados eficazmente com cirurgia e iodo radioactivo, e cerca de 90% dos doentes com menos de 45 anos ficam curados (DeLellis et al., 2004). Por outro lado, os PDTC e os ATC apresentam escassa diferenciação folicular e comportam-se de forma altamente agressiva. Em particular, os ATC apresentam uma progressão clínica muito rápida, e em geral, manifestam no diagnóstico inicial, uma extensa invasão dos tecidos adjacentes e metástases à distância, nomeadamente pulmonares, ósseas ou cerebrais (Muro-Cacho and Ku, 2000; DeLellis et al., 2004). Apesar dos ATC contribuírem apenas para 1 a 2% dos tumores da tiróide (Kondo et al., 2006; Nikiforov and Nikiforova, 2011), são responsáveis por 14 a 50% das mortes relacionadas com cancro da tiróide. Estão associados a uma sobrevivência média de 3 a 5 meses (DeLellis et al., 2004; Nagaiah et al., 2011), representando assim uma das neoplasias mais letais. Os PDTC apresentam características morfológicas e clínicas intermédias entre os WDTC e ATC (Lam et al., 2000; Volante et al., 2004; Sanders et al., 2007; Nambiar et al., 2011). A taxa média de sobrevida aos 5 anos, dos doentes com PDTC é de cerca de 50% e a mortalidade deve-se sobretudo a metástases pulmonares e ósseas (DeLellis et al., 2004; Nambiar et al., 2011). Os PDTC e em especial, os ATC, são refractários às formas convencionais de tratamento (cirurgia e iodo radioactivo). Na maioria dos casos, a ressecção dos tumores não é viável e a quimio- e a radioterapia demonstram um efeito reduzido na sobrevivência dos doentes (Sanders et al., 2007; Smallridge et al., 2009; Abate and Smallridge, 2011). Assim, é de extrema importância a implementação de novas modalidades terapêuticas que sejam eficazes nestas neoplasias. A transformação do epitélio folicular da tiróide resulta tipicamente de alterações genéticas que envolvem componentes das vias de sinalização MAPK-ERK e PI3K-AKT, essenciais na regulação da proliferação e homeostasia celular. Nos PTC detectam-se, de forma mutuamente exclusiva (Kimura et al., 2003; Nikiforov and Nikiforova, 2011), mutações activantes nos genes que codificam a cinase de serina/treonina BRAF e as GTPases H-, K- e NRAS, sendo a mutação do gene BRAF a alteração mais frequente (Kimura et al., 2003; Fugazzola et al., 2006). Entre os WDTC, as mutações nos genes RAS estão associadas a tumores com morfologia folicular, sendo detectadas na variante folicular dos PTC e em FTC (Vasko et al., 2003; Zhu et al., 2003; Di Cristofaro et al., 2006). As características clínicas e histológicas dos carcinomas da tiróide sugerem uma contínua perda da diferenciação celular (um processo de desdiferenciação) pelo qual ocorre um aumento progressivo da agressividade. Considera-se que os PDTC e ATC possam surgir directamente a partir da célula folicular ou derivarem de tumores pré-existentes. Este último modelo é apoiado pela frequente detecção de áreas bem-diferenciadas em casos de PDTC e ATC (Nikiforova et al., 2003a; DeLellis et al., 2004; Quiros et al., 2005; Takano et al., 2007a; Wang et al., 2007a; Santarpia et al., 2008; Ricarte-Filho et al., 2009). Várias evidências moleculares sugerem, de igual modo, que os WDTC podem progredir para PDTC e para ATC. A detecção dos oncogenes RAS e BRAF em casos de PDTC e ATC (Garcia- Rostan et al., 2003; Smallridge et al., 2009; Volante et al., 2009) e a presença destas mutações em componentes diferenciadas e indiferenciadas de um mesmo tumor (Oyama et al., 1995; Nikiforova et al., 2003a; Begum et al., 2004; Cohen et al., 2004; Takano et al., 2007a; Costa et al., 2008; Schwertheim et al., 2009), sugerem um processo de progressão a partir de WDTC. Além disso, estas mutações podem coexistir com alterações no gene supressor de tumores TP53 (Lam et al., 2000; Quiros et al., 2005; Wang et al., 2007), no gene CTNNB1 que codifica o efector β-catenina da via WNT (Garcia-Rostan et al., 2001) e no PIK3CA que codifica a subunidade catalítica da cinase PI3K da via PI3K-AKT (Lam et al., 2000; Garcia-Rostan et al., 2001; Garcia-Rostan et al., 2005; Quiros et al., 2005; Hou et al., 2007a; Wang et al., 2007a; Liu et al., 2008; Santarpia et al., 2008; Ricarte-Filho et al., 2009). Estas alterações, que são detectadas exclusivamente, ou com maior frequência em PDTC e ATC, demonstram, assim, a acumulação e a cooperação de eventos específicos durante a desdiferenciação. Os microRNA (miRNA), que com frequência se encontram desregulados no processo oncogénico (Calin and Croce, 2006), podem apresentar padrões de expressão anómalos nos tumores da tiróide (Nikiforova et al., 2009). Cada um destes RNA, que são não-codificantes, pode estar envolvido na regulação de centenas de transcriptos-alvo, mediando a sua degradação ou a repressão da translacção proteica (Carthew and Sontheimer, 2009). Assim, os miRNA podem constituir potenciais agentes terapêuticos, na medida em que afectam simultaneamente várias vias de sinalização, impedindo os mecanismos compensatórios das células tumorais (Lujambio and Lowe, 2012). Os níveis de expressão dos miRNA numa célula são o resultado tanto da sua biossíntese como da sua degradação. Enquanto que a via de biossíntese se encontra bem caracterizada (Cullen, 2006), o processo de degradação e o tempo de semi-vida dos miRNA, são relativamente desconhecidos. A determinação da estabilidade e a caracterização dos factores que influenciam a degradação destas moléculas, representam, portanto, áreas importantes para a investigação da potencial utilidade terapêutica dos miRNA. A análise de expressão génica global no campo da oncologia demonstrou ser vantajosa no prognóstico e classificação dos tumores, assim como, na identificação de potenciais alvos terapêuticos (Chung et al., 2002; Nevins and Potti, 2007). Esta metodologia tem sido extensivamente utilizada na tiróide, especialmente no estudo dos diferentes subtipos de WDTC (Griffith et al., 2006; Riesco-Eizaguirre and Santisteban, 2007). No caso dos PDTC e ATC, a análise da expressão génica global em tumores primários encontra-se restrita a poucos estudos. A comparação dos perfis de expressão de ATC e PDTC com WDTC e tecido tiroideu normal (TN) permitiu identificar assinaturas moleculares relacionadas com a proliferação e ciclo celular, instabilidade cromossómica, adesão, motilidade celular e perda da função tiróidea (Salvatore et al., 2007; Montero-Conde et al., 2008; Hébrant et al., 2012). Com o objectivo de elucidar os mecanismos envolvidos na progressão e na agressividade dos PDTC e ATC e identificar novos alvos para o tratamento destes tumores, compararam-se os perfis de expressão génica globais de amostras de TN e de WDTC com PDTC e ATC. Tendo em conta os resultados obtidos, procedeu-se a uma extensa pesquisa de mutações em genes envolvidos em processos celulares desregulados nos PDTC e ATC, e correlacionaramse os dados obtidos com os aspectos clínico-patológicos dos doentes. Por fim, o estudo do turnover de RNA através da marcação com um análogo da uridina (4-tiouridina), foi usado para determinar os tempos de semi-vida de miRNA numa linha celular de carcinoma desdiferenciado da tiróide. A comparação da expressão génica global entre 5 PDTC, 19 WDTC e 3 TN, e entre 5 ATC e 4 TN revelou que os PDTC e ATC exibem, em comum, genes desregulados com expressão aumentada associados ao ciclo celular, proliferação celular, segregação cromossómica e ao “checkpoint” do fuso mitótico, enquanto que, os genes sub-expressos estavam principalmente relacionados com a adesão celular. Os ATC apresentaram como eventos específicos (ausentes na análise dos PDTC), o aumento de expressão de componentes da via do TGF-β e a sub-expressão de genes associados à função e metabolismo tiroideu, à morfologia epitelial e às junções celulares. A análise não-supervisionada da semelhança génica entre as amostras e a correlação com a pesquisa de alterações do RAS e BRAF, demonstrou que as variantes foliculares de PTC são os precursores mais prováveis dos PDTC com mutação no gene RAS. Por outro lado, a comparação dos genes diferencialmente expressos em cada tipo de tumor em relação ao tecido normal, permitiu avaliar que os ATC partilhavam maior número de genes com os PTC clássicos do que com os restantes tumores, o qual suporta a provável origem dos ATC a partir dos PTC clássicos. A análise dos genes diferencialmente expressos entre cada tipo de tumor e o TN revelou que 307 de 494 (60%) dos genes eram sobre-expressos em PTC, enquanto que 137 de 171 (80%) estavam subexpressos em FTC, 92 de 107 (86%) estavam sub-expressos em PDTC e 983 de 1333 (74%) estavam com expressão diminuída nos ATC. Validaram-se por RT-PCR quantitativo em grupos independentes de tumores, o gene UHRF1, associado à proliferação e identificado como sobre-expressos nos PDTC em relação ao tecido normal e o gene ITIH5, relacionado com a adesão celular e sub-expresso nos PDTC em relação ao tecido normal. Adicionalmente, caracterizou-se o gene CDKN3, o qual codifica um inibidor das cinases dependentes de ciclina (CDK) que foi identificado, na análise de expressão, como o gene mais sobre-expresso nos ATC, tendo-se confirmado a sua expressão aumentada em ATC, bem como em PDTC relativamente ao TN. De acordo com a sua função supressora de tumor, observou-se uma expressão significativa de formas de splicing aberrantes apenas nos ATC. Devido ao papel importante que a activação da via do TGF-β apresenta na promoção da transição epitelial-mesenquimal (EMT) (Huber et al., 2005), validou-se a expressão do gene SNAI2, regulador do EMT, e cuja expressão é induzida por esta via (Xu et al., 2009). Confirmou-se assim, a sobre-expressão específica deste gene em ATC. Tendo em conta os resultados obtidos nos estudos de expressão génica global dos PDTC e ATC, procedeu-se a uma extensa análise mutacional em genes envolvidos na regulação do ciclo celular como os inibidores de CDK [CDKN1A (p21CIP1), CDKN1B (p27KIP1), CDKN2A (p14ARF, p16INK4A), CDKN2B (p15INK4B) e CDKN2C (p18INK4C)], genes envolvidos na adesão celular (AXIN1, que codifica um regulador negativo da via WNT) e genes cujo envolvimento nestes tumores fora previamente descrito (H-, K-, NRAS, BRAF, PIK3CA, TP53 e CTNNB1). A pesquisa de alterações patogénicas em 26 ATC e 22 PDTC revelou que as mutações dos genes TP53 (presentes em 42% dos ATC e 27% dos PDTC) e RAS (presentes em 31% dos ATC e 18% dos PDTC) são as mais frequentes nestes tumores. As mutações nestes genes apresentaram mútua exclusividade (P=0,0354) e a sua presença estava associada a um menor tempo de sobrevida global dos doentes com PDTC e ATC (P=0,0383). No caso dos inibidores de CDK identificou-se, pela primeira vez, que alterações nos genes CDKN2A e CDKN2B podem estar envolvidos em cerca de 25% dos PDTC. Por outro lado, as mutações previamente descritas como frequentes em ATC, tais como BRAF, PIK3CA, AXIN1 ou CTNNB1 (Smallridge et al., 2009), foram identificadas em menos de 8% dos casos. O método não-disruptivo de marcação de RNA com 4-tiouridina, foi optimizado e utlizado, pela primeira vez, no estudo da estabilidade dos miRNA. A incubação de uma linha celular de tumor desdiferenciado da tiróide (BCPAP), com 200 μM 4-tiouridina durante 24 horas, revelou não ser tóxico para as células, não afectar a expressão dos miRNA e permitiu marcar, purificar e quantificar os miRNA de forma precisa. Recorrendo a “arrays” de expressão de miRNA, obtiveram-se os tempos de semi-vida para 249 miRNA, cujo tempo médio foi de 2,5 dias, variando desde 22 horas (miR-208a e miR-107) até mais de 5,5 dias (miR-1321 e miR-320d). Estes dados revelam que os miRNA apresentam uma maior estabilidade que os RNA mensageiros (Yang et al., 2003; Friedel et al., 2009), sendo comparável à estabilidade proteica (Boisvert et al., 2012). O nosso estudo encontra-se em desenvolvimento mas, pretende-se identificar os factores determinantes para a degradação de miRNA. Em especial, a comparação destes dados com tempos de semi-vida determinados em células normais da tiróide, permitirá identificar miRNA cuja estabilidade se encontra desregulada no processo oncogénico. Em suma, no presente trabalho identificaram-se várias vias e genes que poderão representar alvos para intervenção terapêutica, com impacto no seguimento clínico e na sobrevivência de doentes com tumores agressivos, como são os PDTC e ATC.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/46096/2008); Fundação Calouste Gulbenkian; Chronic Disease Research Centre; Portuguese Society of Endocrinology, Diabetes and Metabolism