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.

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.

A Neoplasia endócrina múltipla tipo 1 (NEM1; OMIM 131100) é uma síndrome endócrina hereditária, que envolve tumores nas glândulas paratireóides, pâncreas endócrino/duodeno e hipófise. Mutações germinativas no gene supressor de tumor MEN1 são identificadas em aproximadamente 80% dos casos familiais. Os casos restantes podem apresentar grandes deleções no gene MEN1 (raras), não identificáveis ao seqüenciamento direto, ou mutações em outros genes, ainda pouco conhecidos. Recentemente, mutações germinativas em genes que codificam quinases dependentes de ciclinas, como o gene supressor de tumor p27Kip1, foram identificadas em cerca de 1-2% dos pacientes NEM1 sem mutação no gene MEN1. Esses pacientes apresentam uma clínica similar à NEM1, sendo chamada de NEM-like ou NEM4. Estudos in vitro mostraram que a proteína codificada pelo gene MEN1, MENIN, controla a expressão gênica de p27Kip1, indicando que ambos os genes fazem parte da mesma via celular supressora de tumor. Devido à correlação genótipo-fenótipo ser muito fraca nessa síndrome e à grande variabilidade fenotípica encontrada em pacientes com NEM1 (mesmo entre indivíduos/familiares que possuem mesma mutação no gene MEN1), no presente estudo investigamos a hipótese do envolvimento do gene p27Kip1, e de outro gene supressor de tumor recentemente associado com um fenótipo tumores hipofisários famílias, o gene AIP, como possíveis moduladores de fenótipo entre os pacientes com NEM1 de uma extensa família brasileira com a mutação germinativa MEN1 c.308delC e ampla variabilidade fenotípica. Dentre uma série de variáveis clínicas investigadas, observamos um possível papel modulador de fenótipo do gene p27Kip1 nesta família com NEM1. Foi encontrada associação significante entre o genótipo do polimorfismo p.V109G do gene p27Kip1, localizado em um domínio de ligação com a proteína p38 (que é um regulador negativo de p27 por levar à degradação dessa proteína), com os seguintes aspectos clínicos: maior agressividade do tumor hipofisário (macro vs. microadenomas), precocidade no desenvolvimento do tumor pancreático, e presença de carcinóides e metástases nos pacientes analisados (p< 0,05). Não foi observada nenhuma associação do gene AIP e o fenótipo dos pacientes com NEM1. O presente estudo investigou, pela primeira vez, o status germinativo do gene p27Kip1 em pacientes com mutação MEN1 e identificou uma associação significante em relação à susceptibilidade e agressividade dos tumores na coorte estudada / Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumoral syndrome that involves tumors in the parathyroids, anterior pituitary and in the pancreatic islet(s) cells. Germline mutations in the tumor suppressor gene MEN1 are detectable through direct sequencing in the majority (80%) of the patients with familial MEN1. The remaining patients may present large MEN1 gene deletions, not detectable through direct sequencing, or mutations in other genes, so far largely unknown. Recently, rare mutations in genes that encode cyclin-dependent kinases, as p27Kip1, have been reported in approximately 1-2% of the patients without a MEN1 mutation. These patients were reported as presenting a MEN1-like (or the MEN4) syndrome phenotype. In vitro studies have demonstrated that the protein encoded by the MEN1 gene, MENIN, controls the expression of the p27Kip1 gene and, therefore, these two genes seem to act in the same intracellular tumor suppressor pathway. Due to the lack of genotype-phenotype correlation in MEN1 and the large clinical variability usually observed within unrelated patients carrying the same MEN1 mutation, we hypothesized that p27Kip1 (as well as AIP gene, recently associated with familial predisposition to pituitary tumors) may act as phenotypic modifying gene(s) in the MEN1 syndrome. Herein, we analyzed possible correlations between p27Kip1 genotype and a number of clinical features. We identified significant statistic associations between the p.V109G p27Kip1 polymorphism and phenotype manifestations, indicating a potential role of p27Kip1 in modifying MEN1 phenotype, as follows: pituitary tumor size; early development of pancreatic tumors, and presence of carcinoids and metastasis (p< 0,05). In addition, a possible association with the AIP gene was excluded. The present study analyzed, for the first time, the germline status of p27Kip1 gene in MEN1-mutated patients and identified a potential interaction between the genotype of this tumor suppressor gene in regulating susceptibility and the tumor aggressiveness in MEN1 patients

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.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

CDC73, also known as HRPT2, is a tumour suppressor gene whose expression is lost or downregulated in parathyroid, renal, breast, uterine and gastric cancers. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. As part of the Paf1 complex, it remains associated with ribonucleic acid (RNA) polymerase II and is involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly(A) length control and, coupling of transcriptional and posttranscriptional events. It has been reported to negatively regulate cellularproliferation by targeting oncogenes CCND1 (cyclin D1) and MYC (c-Myc). Moreover, it has also been indicated to inhibitβ-catenin-mediated transcription. Taken together, these findings strongly suggest that it contributes to the expression of genes whose products have an important role in the suppression of tumor development and cell death. In this study, we have attempted to study the transcriptional and posttranscriptional regulation of CDC73 and its role in OSCC. The main findings of the present study are listed below. 1. To begin with, the expression analysis of CDC73 was performed both at the RNA and the protein levels by qRT-PCR and IHC, respectively. As expected, a majority of the OSCC samples showed downregulation of CDC73 both at the RNA and the protein levels compared to their normal oral tissues. 2. Loss-of-heterozygosity (LOH), mutation and promoter methylation are the hallmarks of a tumor suppressor gene (TSG). Therefore, to characterize CDC73 as a TSG in OSCC and to look into the mechanisms that could be the cause of CDC73 downregulation in OSCC, LOH, mutation and promoter methylation of CDC73 were studied. The results showed that LOH, mutation and promoter methylation are not the major causes of CDC73 downregulation in OSCC. 3. To identify the alternate mechanisms as the cause of CDC73 downregulation in OSCC, a combination of bioinformatics and molecular approaches were used. The results showed that the upregulation of an inhibitory transcription factor WT1 (Wilms tumor protein 1) and an oncogenic microRNA-155 are the major causes of its downregulation in OSCC. 4. The luciferase reporter assay of SCC131 cells co-transfected with a WT1 construct and a CDC73 promoter construct showed that WT1 over expression represses CDC73 expression in a dose-dependent manner. 5. Due to the presence of zinc fingers in its C-terminal half, WT1 has been found to be a potent transcriptional regulator of genes. Therefore, to determine if WT1 down regulates CDC73 via binding its promoter, the chromatin immunoprecipitation (ChIP) assay was performed. The results showed the binding of WT1 to the CDC73 promoter in vivo. Binding of WT1 to the CDC73 promoter was further confirmed in vitro by the electrophoretic mobility shift assay (EMSA). 6. The 5-aza-2’-deoxycytidine (AZA) treatment of SCC131 cells led to upregulation of WT1 with a concomitant downregulation of CDC73. The COBRA technique demonstrated that the upregulation of WT1 upon the 5-AZA treatment was due to its promoter methylation. 7. To determine if the WT1-mediated reduction of CDC73 expression has a functional relevance in cell growth and proliferation, we knocked down CDC73 expression by transient over expression of WT1 in SCC131 cells and quantitated cell proliferation by the MTT assay. As expected, the results demonstrated that the reduced CDC73 level was associated with an increased cell proliferation. Cotransfection of CDC73 with WT1 in SCC131 cells attenuated the pro-oncogenic effect of WT1 by apoptosis induction. 8. After validating CDC73 as the target of WT1 by bioinformatics and in vitro assays, we quantitated the expression levels of WT1 and CDC73 by qRT-PCR in OSCC samples and their matched normal oral tissue samples. The results showed an inverse correlation between the expression levels of WT1 and CDC73 in a majority of the samples. To exclude the possibility of alternate mechanisms as the cause of CDC73 downregulation in OSCC, we selected a subset of OSCC samples with downregulated level of CDC73 and analysed them for LOH at the CDC73 locus and promoter methylation. Further, some of these OSCC samples were also analyzed for mutations in CDC73. The results showed that these OSCC samples did not have LOH, promoter methylation or any mutation, again validating the fact that CDC73 is a biological target of oncogenic WT1, and the transcriptional repression of CDC73 by WT1 could be a major mechanism for CDC73 downregulation in OSCC. 9. Recent studies have shown that a growing class of noncoding RNAs called microRNAs (miRNAs) is involved in posttranscriptional regulation of genes. There is a growing body of literature supporting the potential role of miRNAs in tumorigenesis. The importance of CDC73 in orchestration of several cellular functions and its role in tumorigenesis make it an attractive candidate for miRNA-mediated regulation of cell growth and proliferation. Using bioinformatics approaches, we identified an oncogenic microRNA-155 (miR-155) that could posttranscriptionally regulate CDC73 expression. 10. Consistent with its oncogenic role, miR-155 was found dramatically upregulated in OSCC samples and was found to be another mechanism for downregulation of CDC73 in a panel of human cell lines and a subset of OSCC samples in the absence of LOH, mutations and promoter methylation. 11. miRNAs regulate posttranscriptional gene expression generally via binding to their cognate sites in the 3’UTR. Therefore, a luciferase reporter construct was made by cloning the 3’UTR of CDC73 downstream to the luciferase reporter gene and the reporter assay was performed. Our experiments clearly indicated that the mature miR-155 regulates CDC73 expression by interacting with its 3’UTR in a site specific manner. 12 Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells over expression miR-155 resulted in increased CDC73 level, decreased cell viability, increased apoptosis and marked regression of engrafts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3’UTR in CDC73. In nutshell, the knockdown of CDC73 expression due to over expression of WT1 and miR-155 not only adds a novelty to the list of mechanisms responsible for its downregulation in different tumors, but the restoration of CDC73 levels by the use of inhibitors to WT1 and antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.