O diagnóstico precoce dos Segundos Tumores Primários (STP) em pacientes já tratados por um carcinoma de cabeça e pescoço deve ser realizado, pois possibilita um tratamento resolutivo com baixa morbidade. O objetivo deste trabalho é avaliar se a cromoscopia com Lugol permite uma melhora na identificação de lesões malignas ou pré malignas em fases iniciais na boca e orofaringe, bem como se a imunoexpressão do p53 e da Metalotioneína no tumor índice predizem o aparecimento de um STP. Foi realizado um estudo prospectivo onde dois grupos comparáveis de portadores de carcinoma epidermóide de cabeça e pescoço foram formados (um com 106 pacientes e outro com 105 pacientes). Foram acompanhados durante um período médio de 25 meses aproximadamente. No primeiro grupo (grupo A) não foram utilizados corantes, já no segundo (grupo B) utilizou-se o Lugol. Foi observado um número de diagnósticos 200% maior no grupo em que foi utilizada a coloração de Lugol (grupo B) em relação ao grupo A. A imunoexpressão aumentada do p53 no tumor índice foi estatisticamente significante quando o paciente desenvolveu um segundo tumor primário diagnosticado pelo Lugol, não visível sem o corante, o que não ocorreu com a metalotioneína. / The early diagnosis of seconds primary tumors (STP) in patients already treated for carcinoma of the head and neck should be done, because enables a resolutive treatment with low morbidity. The objective of this paper is to evaluate whether chromoscopia using Lugols solution allows an improvement in the identification of malignant or pre malignant lesions in early stages in the mouth and oropharynx, and whether the expression of P53 and metallothionein in tumor index predict the emergence of a STP. A prospective study was conducted, where two groups statistically similar of patients with head and neck squamous cell carcinoma (one with 106 patients and another with 105 patients) were followed-up for a median period of 25 months, approximately. In the first group dyes were not used, and in the second Lugols solution was employed. It was observed a number of diagnoses 100%i higher in the group that Lugols solution was used. The increasing of P53 expression in tumor index was statistically significant when the patient developed a second primary tumor diagnosed by Lugol, not visible without dye, which has not occurred with metallothionein.

Poon, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-124). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.6 / Chapter Chapter 2 --- Literature Reviews --- p.8 / Chapter 2.1 --- Cancer genetics --- p.8 / Chapter 2.1.1 --- Oncogenes and TSGs --- p.8 / Chapter 2.1.2 --- Kundsońةs two-hit event of cancer gene --- p.9 / Chapter 2.2 --- Cancer Epigenetics --- p.9 / Chapter 2.2.1 --- Types of Epigenetic regulation --- p.10 / Chapter 2.2.2 --- DNA methylation in TSGs --- p.10 / Chapter 2.2.2.1 --- Promoter CpG island in DNA methylation --- p.10 / Chapter 2.2.2.2 --- Protection system in DNA methylation --- p.11 / Chapter 2.2.2.3 --- Transcriptional silencing by DNA methylation --- p.11 / Chapter 2.2.2.4 --- DNA methylation of TSG silencing in cancers --- p.13 / Chapter 2.2.3 --- Hypomethylation of the cancer genome --- p.14 / Chapter 2.2.4 --- Clinical relevance of cancer epigenetic --- p.14 / Chapter 2.3 --- EGFR/STAT/SOCS pathway --- p.15 / Chapter 2.3.1 --- General Introduction of the EGFR pathway --- p.15 / Chapter 2.3.2 --- EGFR survival signaling pathways --- p.16 / Chapter 2.3.3 --- EGFR/STAT/SOCS signaling --- p.17 / Chapter 2.3.4 --- EGFR/STAT/SOCS signaling and cancers --- p.18 / Chapter 2.3.4.1 --- EGF and cancers --- p.18 / Chapter 2.3.4.2 --- EGFR/STAT/SOCS pathway and cancers --- p.18 / Chapter 2.3.4.3 --- EGF survival signaling as a target for cancer therapy --- p.19 / Chapter 2.4 --- TSGs in the EGFR/STAT/SOCS pathway --- p.20 / Chapter 2.4.1 --- Suppressors of cytokine signaling (SOCS) family --- p.20 / Chapter 2.4.2 --- Signal transducers and activators of transcription (STATs) family --- p.22 / Chapter 2.4.3 --- Sprouty (SPRY) family --- p.23 / Chapter 2.4.4 --- Protein Inhibitor of Activated STAT (PIASs) family --- p.25 / Chapter 2.4.5 --- Ras and Rab Interactor (RIN) family --- p.26 / Chapter 2.4.6 --- Ras-association domain family (RASSF) --- p.26 / Chapter 2.4.7 --- Glycine N-methyltransferase (GNMT) --- p.28 / Chapter 2.5 --- Nasopharyngeal carcinoma (NPC) --- p.30 / Chapter 2.5.1 --- Epidemiology of NPC --- p.30 / Chapter 2.5.2 --- Histopathology of NPC --- p.30 / Chapter 2.5.3 --- Genetic and epigenetic alteration in NPC --- p.31 / Chapter 2.5.4 --- EGFR signaling in NPC --- p.32 / Chapter 2.6 --- Esophageal squamous cell carcinoma (ESCC) --- p.33 / Chapter 2.6.1 --- Epidemiology of ESCC --- p.34 / Chapter 2.6.2 --- Histopathology of ESCC --- p.34 / Chapter 2.6.3 --- Genetic and epigenetic alteration in ESCC --- p.35 / Chapter 2.6.4 --- EGFR signaling in ESCC --- p.36 / Chapter Chapter 3 --- Materials and Methods --- p.38 / Chapter 3.1 --- General Materials --- p.38 / Chapter 3.1.1 --- "Cell lines, tumor and normal tissue samples" --- p.38 / Chapter 3.1.2 --- Maintenance of cell lines --- p.38 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.39 / Chapter 3.1.4 --- Total RNA extraction --- p.39 / Chapter 3.1.5 --- Genomic DNA extraction --- p.40 / Chapter 3.2 --- General techniques --- p.40 / Chapter 3.2.1 --- Agarose gel electrophoresis of DNA --- p.40 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.40 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.41 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.41 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.41 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.42 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.42 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.42 / Chapter 3.3 --- Preparation of reagents and medium --- p.43 / Chapter 3.4 --- Semi-quatitative Reverse-Transcription (RT) PCR expression analysis --- p.44 / Chapter 3.4.1 --- Reverse transcriptin reaction --- p.44 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.44 / Chapter 3.4.2.1 --- Primers design --- p.44 / Chapter 3.4.2.2 --- PCR reaction --- p.46 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.47 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.47 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.48 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.48 / Chapter 3.5.2.2 --- Primers design --- p.48 / Chapter 3.5.2.3 --- PCR reaction --- p.49 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.50 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.51 / Chapter 3.6.1 --- Sub-cloning of expression vector of candidate genes --- p.51 / Chapter 3.6.1.1 --- Mouse Socsl expression vector --- p.51 / Chapter 3.6.1.2 --- SPRY1 expression vector --- p.51 / Chapter 3.6.1.3 --- GNMT expression vector --- p.52 / Chapter 3.6.2 --- Restriction digestion of cloning vectors and expression --- p.52 / Chapter 3.6.3 --- Ligation of cloning fragments --- p.53 / Chapter 3.6.4 --- Colony formation assay on monolayer culture --- p.53 / Chapter 3.6.5 --- Statistical analysis --- p.54 / Chapter Chapter 4 --- Screening of candidate TSGs in EGFR pathway --- p.55 / Chapter 5.3.3 --- Restoration of GNMT expression by pharmacological demethylation --- p.89 / Chapter 5.3.4 --- Confirmation of the methylation status of GNMT promoter by BGS --- p.90 / Chapter 5.3.5 --- Methylation status of GNMT in ESCC and NPC primary tumors --- p.90 / Chapter 5.3.6 --- GNMT inhibited the growth of tumor cells in-vitro --- p.90 / Chapter 5.3.7 --- Discussion --- p.95 / Chapter Chapter 6 --- General Discussion --- p.100 / Chapter Chapter 7 --- Summary --- p.105 / Chapter Chapter 8 --- Future Study --- p.107 / Reference --- p.109

As muitas formas diferentes de câncer representam uma grande dimensão no âmbito da saúde pública mundial. Embora os esforços da medicina diagnóstica, vários tumores permanecem sem resposta à terapia tradicional. Uma alternativa é o uso de terapia gênica, a qual consiste a transferência de um gene terapêutico para a célula tumoral com a expectativa de inibição da progressão tumoral. Nosso laboratório desenvolveu uma série de vetores adenovirais onde a expressão do transgene é controlada pela p53 e usamos esses vetores para mostrar que a presença de p19Arf (um parceiro funcional de p53) sensibiliza células de melanoma murino, B16-F10 (p53-tipo selvagem), associado à ação do interferão-beta (IFNbeta, uma citocina pleiotrópica) quando testado in vitro. Mesmo que os vetores adenovirais representem o sistema de transferência gênica mais utilizado para a terapia de genes de câncer, seu uso por via sistêmica é limitado principalmente por inativação pelo sistema imune. Diferentes técnicas visam proteger as partículas de vírus do sistema imunológico e direcioná-las para o leito tumoral. Uma dessas técnicas envolve a utilização de células estaminais mesenquimais (MSCs). As propriedades dos MSC incluem a auto renovação, o potencial de diferenciação, bem como a sua capacidade de migrar e infiltrar tumores. Para este fim, nosso objetivo era utilizar MSCs murinos como portadores de adenovírus que expressam IFNbeta e para verificar se a presença de p19Arf nas células tumorais aumentaria a sua sensibilidade para IFNbeta. Para itso, os CTMs foram isolados da medula óssea ou do tecido adiposo de ratinhos C57BL/ 6 machos. Foi verificada a presença de marcadores de CTM (Sca1, CD29) e a ausência de marcadores para linhagens hematopoiéticas (CD31, CD11b, CD45). Sendo as CTM do tecido adiposo foram mais fáceis de cultivar, estes foram utilizados nos seguintes ensaios. In vitro, a aplicação do vector adenoviral que codifica um gene repórter (eGFP) resultou em mais de 70% de eficiênciamde transdução de CTM, sem indução de alterações morfológicas até 72 horas após o tratamento. A aplicação de vector portador de IFNbeta também foi bem tolerada, no entanto transdução com p19Arf sozinho ou em combinação com IFNbeta induziu alterações morfológicas nas CTMs. Em seguida, as células B16-F10 foram transduzidas ou não com o vetor codificando p19Arf e co-cultivadas com MSCs que foram transduzidas ou não com IFNbeta, demonstrando que a presença de p19Arf confere sensibilidade aumentada de células B16-F10 ao tratamento com IFNbeta . Em ensaios preliminares, os tumores B16-F10 foram estabelecidos subcutaneamente em camundongos C57BL / 6 e, posteriormente, as MSC marcadas com eGFP foram aplicadas na circulação após a injeção da veia da cauda. Após 48 horas, estes tumores foram recuperados e a presença de células positivas para eGFP foi confirmada, indicando que os MSCs se infiltraram no microambiente do tumor / The many different forms of cancer represent a tremendous investment for public health all over the world. Although the efforts of both diagnostic and therapeutic medicine have reduced the number of deaths due to cancer, many tumor types remain impervious to traditional therapy. An alternative is the use of gene therapy which entails the transfer of a therapeutic gene to the tumor cells with the expectation of inhibiting tumor progression. Our laboratory has developed a series of adenoviral vectors where transgene expression is controlled by p53 and we have used these vectors to show that the presence of p19Arf (a functional partner of p53) sensitizes murine melanoma cells, B16-F10 (p53-wild type), to the action of interferon-beta (IFNbeta, a pleiotropic cytokine) when tested in vitro. Even though adenoviral vectors are the most utilized gene transfer system for cancer gene therapy, their systemic application is limited principally by immune inactivation. Different techniques aim to protect the virus particles from the immune system and to direct them to the tumor bed. One of these techniques involves the utilization of mesenchymal stem cells (MSCs). The properties of MSCs include self-renewal, the potential for differentiation as well as their ability to migrate to and infiltrate tumors. To this end, our objective was to utilize murine MSCs as carriers of adenovirus that express IFNbeta and to verify if the presence of p19Arf in the tumor cells would enhance their sensitivity to IFNbeta. For this, MSCs were isolated from bone marrow or adipose tissue from male C57BL/6 mice. The presence of MSC markers (Sca1, CD29) was verified as was the absence of markers for hematopoietic lineages (CD31, CD11b, CD45). Since the MSCs from adipose tissue were easier to cultivate, these were utilized in the following assays. In vitro, application of the adenoviral vector encoding a reporter gene (eGFP) at a multiplicity of infection of 1000 resulted in the transduction of more than 70% of the MSCs and without the induction of morphological alterations even by 72 hours post treatment. The application of a vector encoding IFN? was also well tolerated, however transduction with p19Arf alone or in combination with IFNbeta induced morphologic alterations in the MSCs. Next, B16-F10 cells were transduced or not with the vector encoding p19Arf and co-cultivated with MSCs that had been transduced or not with IFNbeta, demonstrating that the presence of p19Arf confers enhanced sensitivity of B16-F10 cells to the treatment with IFN?. In preliminary assays, B16-F10 tumors were established subcutaneously in C57BL/6 mice and later MSCs labeled with eGFP were applied in the circulation upon tail vein injection. After 48 hours, these tumors were recovered and the presence of eGFP-positive cells was confirmed, indicating that the MSCs infiltrated the tumor microenvironment

A acromegalia é doença insidiosa e desfigurante caracterizada por um crescimento desproporcional dos ossos das mãos, pés e do crânio devido à exposição crônica a altos níveis de hormônio de crescimento (GH) e de seu efetor insuline growth factor 1 (IGF-1). Trata-se de uma doença rara, com incidência estimada de 3-4 casos por milhão, com prevalência de aproximadamente 50 casos por milhão de pessoas. A principal causa da acromegalia é a presença de um tumor hipofisário secretor de GH (somatotropinoma). Caso o somatotropinoma ocorra durante a infância ou adolescência, antes do fechamento das epífises dos ossos longos, a criança crescerá longitudinalmente de forma descontrolada, caracterizando a forma clínica gigantismo. Na grande maioria dos casos a acromegalia se apresenta na forma esporádica, entretanto casos familiais da doença podem ocorrer associados à Neoplasia Endócrina Múltipla tipo 1 (NEM-1), ao complexo de Carney (CNC) e à acromegalia familial isolada (IFS). Os genes responsáveis pela NEM-1 (MEN1) e CNC (PRKAR1A) foram clonados há mais 10 anos, entretanto etiologia molecular da IFS permaneceu desconhecida até recentemente. Vierimaa et al. (2006) combinaram estudos de ligação por análise de polimorfismos e estudos de expressão gênica e identificaram mutações no gene AIP em famílias com acromegalia não-NEM-1 e não-CNC; além de perda de heterozigose (LOH) nos somatotropinomas dos pacientes com mutação AIP. No presente estudo, investigamos o gene AIP em três famílias brasileiras com IFS e em seus tumores (hipofisários e não-hipofisários). Descrevemos uma nova mutação AIP (Y268X) em uma família brasileira com IFS, confirmando o papel desse novo gene na predisposição a tumores hipofisários. A partir de dados gerados em uma extensa revisão da literatura, sugerimos que os tumores hipofisários familiais isolados são doenças multigênicas que possuiriam um gene principal, mas que sofreriam influência de outros genes/loci ainda pouco caracterizados. Assim, investigamos também o envolvimento de diversos genes/loci candidatos (SSTR2, SSTR5, CDKN1B, AHR, PRKAR1A, PTTG, PROP1, MEG3, RB1 e 2p16) como possíveis moduladores do fenótipo na IFS. Nossos dados sugerem que além da mutação AIP, há necessidade da co-segregação de marcadores localizados em regiões com potencial oncogênico para o desenvolvimento da doença hipofisária. Também apresentamos nesta Tese as primeiras análises de tumores nãohipofisários em pacientes com mutação AIP e encontramos evidências do possível envolvimento de AIP na tumorigênese de um carcinoma funcionante do córtex adrenal de paciente com IFS. / Acromegaly is a rare disfigurating and insidious disease characterized by enlargement of hands, feet and skull bones due to excess of growth hormone (GH) secreted by a pituitary tumor (somatotropinoma). The majority of the cases with acromegaly is sporadic, however it may occur in association with inherited disorders as Multiple Endocrine Neoplasia type 1 (MEN1), Carney complex (CNC) and Isolated Familial Somatotropinoma (IFS). The genes associated with MEN1 syndrome (MEN1) and CNC (PRKAR1A) have been described more than a decade ago, however until very recently the molecular etiology of IFS remained unknown. Using a combined strategy of single nucleotide polymorphism (SNP) analysis and gene expression analysis, Vierimaa et al. (2006) described mutations in the AIP gene occurring in families with acromegaly not associated with MEN1 and CNC. In the current study, we investigated three Brazilian families with IFS and were able to describe two germline mutations in the AIP gene, confirming the role of this new gene in the predisposition to familial somatotropinoma. We revised the literature of genetic studies of isolated pituitary adenoma syndromes, which indicated a genetic heterogeneity as well as possible multigenic inheritance for these diseases. Thus, we investigated the role of several genes/loci (SSTR2, SSTR5, CDKN1B, AHR, PRKAR1A, PTTG, PROP1, MEG3, RB1 and 2p16) selected as potentially acting as phenotypic modulators in IFS. Our data indicate that AIP-mutated patients are prone to pituitary disease, however it is necessary the co-segregation of markers located at oncogenic regions to the development of the pituitary tumors and manifestation of the disease. Herein, we also present the first somatic analysis of non-pituitary tumors of AIP-mutated patients. A potential role of AIP, which is implicated in the cAMP pathway, could not be excluded in the development of an adrenocortical carcinoma.

Les gènes ING (Inhibitor of Growth) sont des gènes candidats suppresseurs de tumeurs conservés de la Levure à l'Homme. Les protéines ING ont des fonctions suppressives de tumeurs de type I ou « caretaker » car elles participent aux processus de maintien de la stabilité du génome en régulant la réplication et la réparation de l'ADN. Elles ont aussi des fonctions suppressives de tumeurs de type II ou « gatekeeper » puisqu'elles sont impliquées dans la régulation de la prolifération cellulaire de façon dépendante et indépendante de p53 et car elles contrôlent la transcription génique en participant au remodelage de la chromatine. L'objectif de ma thèse est de mieux comprendre l'implication de ING1, ING2 et ING3 dans les voies de suppression des tumeurs. Nos travaux montrent que ING1 est sumoylée sur la lysine 193 principalement par l'E3 SUMO ligase PIAS4, afin de réguler l'ancrage de ING1 sur le promoteur de gènes cibles pour réguler leur transcription. Nous avons aussi décrit pour la première fois l'implication de ING2 et de ING3 dans la réponse aux cassures double brin de l'ADN. Nous montrons que cette fonction est conservée entre ING2, ING3 et leur orthologues, respectivement, Pho23 et Yng2 chez la Levure Saccharomyces cerevisiae. ING2 contrôle l'accumulation de PIAS4 au niveau des sites de dommages et régule la sumoylation de l'E3 ubquitine ligase RNF168, afin de permettre la signalisation et la réparation des cassures double brin de l'ADN. ING3 est nécessaire à l'accumulation de 53BP1 et contrôle la réparation de ces dommages. Ces travaux contribuent donc à une meilleure connaissance du rôle des ING dans les voies de suppression des tumeurs. Ils permettent de mieux comprendre comment ING1 régule la transcription génique et décrivent une nouvelle fonction suppressive de tumeur de type I ou « caretaker » pour ING2 et ING3 dans le maintien de la stabilité du génome. / ING (Inhibitor of Growth) genes are tumor suppressor gene candidates conserved from Yeast to Humans. ING proteins have type I tumor suppressive functions or "caretaker" because they participate in the maintenance of genome stability by regulating DNA replication and repair processes. They have also tumor suppressive functions of type II or "gatekeeper" because they are involved in the regulation of cell proliferation in p53 dependent and independent manners. They also participate in the regulation of gene transcription by regulating chromatin remodeling. The aim of my thesis was to better understand how ING1, ING2 and ING3 are involved in tumor suppressive pathways. Our work shows that ING1 is sumoylated on lysine 193 mainly by the SUMO E3 ligase PIAS4 to regulate ING1 anchoring on target gene promoters to control gene transcription. We have also described the involvement of ING2 and ING3 in the DNA double strand breaks response. We show the conservation of this function between ING2, ING3 and their orthologs, respectively, Pho23 and Yng2 in Yeast Saccharomyces cerevisiae. ING2 controls the accumulation of PIAS4 at DNA damage sites and regulates the sumoylation of the E3 ubiquitin ligase RNF168, to regulate DNA double strand break signaling and repair. ING3 is necessary for the accumulation of 53BP1 and promotes DNA damage repair. This work contributes to a better understanding of the role of ING proteins in tumor suppression. It thus provides new insights of how ING1 regulates gene transcription and emphasizes a new tumor suppressive function of type I or "caretaker" for ING2 and ING3 in the genome stability maintenance.

Copy number variation (CNV) comprises a recently discovered kind of variation involving deletion and duplication of DNA segments of variable size, ranging from a few hundred basepairs to several million. By altering gene dosage levels or disrupting proximal or distant regulatory elements CNVs create human diversity. They represent also an important factor in human evolution and play a role in many disorders including cancer. Array-based comparative genomic hybridization as well as expression arrays are powerful and suitable methods for determination of copy number variations or gene expression changes in the human genome. In paper I we established a 32K clone-based genomic array, covering 99% of the current assembly of the human genome with high resolution and applied it in the profiling of 71 healthy individuals from three ethnic groups. Novel and previously reported CNVs, involving ~3.5% of the genome, were identified. Interestingly, 87% of the detected CNV regions overlapped with known genes indicating that they probably have phenotypic consequences. In papers II through IV we applied this platform to different tumor types, namely two collections of brain tumors, glioblastoma (paper II) and medulloblastoma (paper III), and a set of bladder carcinoma (paper IV) to identify chromosomal alterations at the level of DNA copy number that could be related to tumor initiation/progression. Tumors of the central nervous system represent a heterogeneous group of both benign and malignant neoplasms that affect both children and adults. Glioblastoma and medulloblastoma are two malignant forms. Glioblastoma often affects adults while the embryonal tumor medulloblastoma is the most common malignant brain tumor among children. The detailed profiling of 78 glioblastomas, allowed us to identify a complex pattern of aberrations including frequent and high copy number amplicons (detected in 79% of samples) as well as a number of homozygously deleted loci. These regions encompassed not only previously reported oncogenes and tumor suppressor genes but also numerous novel genes. In paper III, a subset of 26 medulloblastomas was analyzed using the same genomic array. We observed that alterations involving chromosome 17, especially isochromosome 17q, were the most common genomic aberrations in this tumor type, but copy number alterations involving other chromosomes: 1, 7 and 8 were also frequent. Focal amplifications, on chromosome 1 and 3, not previously described, were also detected. These loci may encompass novel genes involved in medulloblastoma development. In paper IV we examined for the presence of DNA copy number alterations and their effect on gene expression in a subset of 21 well-characterized Ta bladder carcinomas, selected for the presence or absence of recurrences. We identified a number of novel genes as well as a significant association between amplifications and high-grade and recurrent tumors which might be clinically useful. The results derived from these studies increase our understanding of the genetic alterations leading to the development of these tumor forms and point out candidate genes that may be used in future as targets for new diagnostic and therapeutic strategies.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Made available in DSpace on 2015-07-22T20:50:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-29. Added 1 bitstream(s) on 2015-08-11T03:25:24Z : No. of bitstreams: 1 Publico-00211a.pdf: 1874263 bytes, checksum: 9fa88ecbdc678f5d003c91ff7ce39eca (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:25Z : No. of bitstreams: 2 Publico-00211a.pdf: 1874263 bytes, checksum: 9fa88ecbdc678f5d003c91ff7ce39eca (MD5) Publico-00211b.pdf: 1651291 bytes, checksum: 797f8caf18af70f6f0cf21a1510830fa (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:25Z : No. of bitstreams: 3 Publico-00211a.pdf: 1874263 bytes, checksum: 9fa88ecbdc678f5d003c91ff7ce39eca (MD5) Publico-00211b.pdf: 1651291 bytes, checksum: 797f8caf18af70f6f0cf21a1510830fa (MD5) Publico-00211c.pdf: 1136167 bytes, checksum: b8d06cb6386ddbf8bce0ebd5b67f3d7a (MD5) / Objetivo: Estudar a imunoexpressão das proteínas metaloproteinase-1, metaloproteinase-7 e p53 em pacientes portadores de adenocarcinoma colorretal, correlacionando com os fatores prognósticos clínico-patológicos. Métodos: Foram analisados tecidos fixados em formol e dispostos em blocos de parafina dos tumores de 82 pacientes, por imunohistoquímica, pelo método da estreptavidina-biotina, usando-se a técnica de arranjo em matriz de amostras teciduais (tissue microarray). Na avaliação da positividade dos marcadores foi utilizado um escore categórico, que predeterminou o valor de corte na percentagem de células coradas do tumor. As expressões teciduais das proteínas foram correlacionadas com as varáveis representadas pelo grau de diferenciação celular, estadiamento, tempo livre de doença, recidiva, sobrevida e mortalidade específica. Foram empregados os testes do qui-quadrado e de Kaplan-Meier para verificar as associações dos marcadores com as varáveis estudadas. Para testar a significância das diferenças entre as curvas do tempo livre de doença e da sobrevida foram utilizados os testes de Log-Rank e Wilcoxon. Resultados: A metaloproteinase-1 foi positiva em todos os tumores. A metaloproteinase-7 foi positiva em 50 (61%) e negativa em 32 (39%) tumores. A p53 foi positiva em 70 (85,4%) e negativa em 12 (14,6%) tumores. A correlação da imunoexpressão dos marcadores realizada separadamente e em conjunto não apresentou associação comsignificância estatística com nenhuma das variáveis analisadas. Conclusão: Não houve correlação da associação entre a imunoexpressão das proteínas metaloproteinase-1, metaloproteinase-7 e p53 com a recidiva tumoral, mortalidade, intervalo de tempo livre de doença, sobrevida, grau de diferenciação celular e estadiamento do câncer colorretal. / Purpose: To analyse the immunoexpression of metalloproteinase-1, metalloproteinase-7 and also the p53 in colorectal adenocarcinoma and correlate it with the clinical pathological prognostic factors. Methods: Formalin-fixed, wax-embedded tissue of the tumours of 82 patients were analysed by immunohystochemistry through the streptavidin-biotin method, using the tissue microarray technique. When evaluating the positivism of the markers a categorical score was used, which predetermined the cutting value on the percentage of the tumour’s cored cells. The protein’s tissue expressions were correlated with the variation represented by the degree of cellular differential, stage, relapse-free survival, recurrence, survival and specific mortality. The tests of the qui-square and Kaplan-Meyer were applied with the aim to verify the markers association with the studied variations. In order to test the significance of the differences between the curves of relapse-free survival and of the overall survival, the tests of Log-Rank and Wilcoxon were utilized. Results: The metalloproteinase-1 was found to be positive in all the tumours. The metalloproteinase-7 was found to be positive in 50 (61%) and negative in 32 (39%) of the tumours. The p53 was found to be positive in 70 (85.4%) and negative in 12 (14.6 %) of the tumours. The correlation of the markers expressions separately and in conjunction produced any significant statistical data. Conclusion: There was no correlation between immunoexpression of metalloproteinase-1, metalloproiteinase-7 and p53 with recurrence, mortality, relapse-free survival, survival, degree of cellular differential and stage in colorectal cancer. / TEDE / BV UNIFESP: Teses e dissertações