Dissertations / Theses on the topic 'EBV cancers'

Latent membrane protein 1 (LMP1) is the primary oncogene of Epstein-Barr virus (EBV), which is associated with various malignancies including nasopharyngeal carcinoma (NPC) – a tumour of epithelial origin. The NPC tissues are so heavily infiltrated with leukocytes that the tumour is sometimes also called lympho-epithelioma. In order to understand the mechanisms involved in LMP1 induced carcinogenesis (and inflammation-associated carcinogenesis in general), our lab has generated transgenic mice expressing an NPC variant of LMP1 (LMP1CAO) in the epidermis. LMP1 induced signalling pathways are activated in the pathological tissue, including NFκB, JNK, MAPK and p38 MAPK. In addition multiple proteins involved in proliferation and inflammation are upregulated including EGFR and its ligands, VEGF, and MMP9 amongst others. The skin of these mice, particularly the ears, develops a progressive inflammatory pathology from birth, initiating with hypervascularization (Stage1 (St1)) and hyperplasia (St2), increasing inflammation, leading to necrosis (St3), ulceration (St4), keratocanthoma (St5) and occasional carcinoma formation. The transgenic model permits an analysis of chronic inflammation as it leads to carcinoma, and the important factors involved in this. This thesis describes the experiments and the pre-clinical trials conducted to dissect the inflamed milieu internal of the pre-neoplastic transgenic tissue, with a focus on the immune cells, immune response, autoantigens, danger signals, oxidative stress and perturbed metabolic pathways. The transgenic tissue showed increased infiltration of mast cells, helper T-cells (CD4+), cytotoxic T-cells (CD8+/GranzymeB+) and Treg cells (CD4+/CD25+/FoxP3+). Inhibition of leukocyte recruitment using in vivo L-selectin inhibition not only resulted in slowed progression of the pathology but also could reverse the phenotype, suggesting a prognostic as well as therapeutic potential of L-selectin inhibition. Several inflammatory markers and cytokines, including STAT3, s100A9, CD30, CD30L, L-selectin, IL-3 and IP-10, were upregulated in the transgenic tissue compared to the controls. The role of a pro-inflammatory environment in the progression of the LMP1CAO induced pathology was investigated further by genetic removal of a chemokine decoy receptor D6 that resulted in accelerated progression of the pre-neoplastic as well as neoplastic pathology. Antibody deposition is a feature of this pathology. In order to identify the antigenic targets, various proteomic techniques were used. Immunoprecipitation experiments showed that the chitinase like proteins (CLPs) specifically Chi3L1, Chi3L3 and Chi3L4, are autoantigens in the LMP1CAO induced inflammation. Western blot and IHC analyses of the LMP1CAO tissue revealed that CLPs are expressed at an early stage of inflammation while active chitinases are expressed at the later stages. Moreover, an attempt was made to investigate the sera and biopsies from NPC patients for the expression and secretion of human CLPs, CHI3L1 and CHI3L2. In another approach, the T-cell/B-cell interaction (and thus Ig class switching) was disrupted through genetic elimination of CD40. The LMP1CAO/CD40KO mice showed delayed progression of the inflammatory phenotype during later stages, suggesting that Ig deposition is factorial in this process. Metabolic fingerprinting of the LMP1CAO tissue revealed deregulation of several metabolic pathways and suggested that “metabolic demand precedes the increased supply during pre-neoplasia”. Several metabolic changes indicative of increased cell proliferation were identified in the transgenic tissue, including upregulation of phospholipid metabolism intermediates, such as choline, phosphocholine, glycerophosphocholines and lysophosphatidic acid. A tumour specific glycolytic programme was operational in the transgenic tissue along with increased levels of glutamine, suggesting tumour-like energy consumption. The transgenic tissue was under oxidative stress indicated by increased levels of H2O2. Finally, in vivo administration of an anti-oxidant, N-acetylcysteine (NAC), arrested the inflammatory phenotype at very early stages, suggesting that oxidative stress is one of the inciting dangers/damages in the LMP1CAO induced inflammation. To summarize, the investigations presented herein have identified several candidates of diagnostic and therapeutic potential that are worth exploring further; not only in relation to the LMP1CAO induced carcinogenesis but also for various cancers in general.

The etiology of nasopharyngeal carcinoma (NPC) is based on intricate interactions among environmental factors, genetic susceptibility and Epstein-Barr virus (EBV) infection. Information concerning the role of EBV infection, particularly during the early stage of NPC development is poorly understood. Our laboratory has shown that stable infection of EBV could be achieved in immortalized epithelial cell lines which harbor genetic alterations and altered cell signaling pathway. In this study, these cell models were used to elucidate early events involved in EBV infection in premalignant nasopharyngeal epithelial cell models and their implications on development and progression of nasopharyngeal carcinoma. The response of EBV-infected cells to a stromal inflammatory cytokine, interleukin-6 (IL-6), was examined. EBV infection and long-term propagation of EBV-infected nasopharyngeal epithelial cells confer enhanced sensitivity to STAT3 activation induced by IL-6. IL-6-induced STAT3 activation reinforced their malignant properties in nasopharyngeal epithelial cells and may play a role in the development of nasopharyngeal carcinoma. Furthermore, constitutive STAT3 activation was demonstrated to facilitate malignant transformation of EBV-infected premalignant nasopharyngeal epithelial cells to cancer cells, suggesting that EBV infection and STAT3 activation might synergistically promote the development of NPC. This study also provides support for the existence of a positive feedback loop of IL-6/STAT3/LMP in NP460hTert-EBV cells, which enhanced STAT3 activation in EBV-infected cells. Elevated levels of IL-6Rα expression were observed in EBV-infected NP460hTert cells compared with uninfected cells and were largely responsible for the enhanced sensitivity of IL-6-induced STAT3 activation in these cells. High expression level of IL-6Rα could amplify IL-6 signaling in nasopharyngeal epithelial cells to promote growth proliferation in NP460hTert cells and increase the growth rate of xenografted NPC cells in immune-suppressed animals, suggesting that IL-6Rα overexpression may play a role of contributing to the development of nasopharyngeal carcinoma. The serum concentrations of both IL-6 and sIL-6R were also higher in NPC patients than healthy individuals and may have prognostic values to predict clinical outcome and disease progression in NPC patients. In conclusion, these data support the hypothesis that EBV infection under inflammatory environment may activate aberrant gene expressions and cell signaling to facilitate malignant transformation. The inflammatory cytokine, IL-6, may mediate the role of EBV infection in the development of NPC.
published_or_final_version
Anatomy
Doctoral
Doctor of Philosophy

En pathologie tumorale, l’étude du micro-environnement tumoral doit prendre en compte différents modes de communication cellulaire : contacts directs entre membranes plasmiques, émission et réception de cytokines et enfin émission et internalisation d’objets biologiques plus complexes comme les microvésicules et les exosomes qui peuvent être assimilés à de véritables organites extra-cellulaires. Le virus d’Epstein-Barr (EBV) participe à l’oncogenèse de plusieurs affections malignes humaines d’origine épithéliale (carcinomes nasopharyngés ou NPC) ou lymphocytaire (lymphomes post-transplantation). Dans ces tumeurs, les cellules malignes qui sont infectées de façon latente par EBV libèrent des exosomes et des microvésicules qui contiennent des protéines et des acides nucléiques d’origine virale. L’étude de ces éléments doit permettre de mieux comprendre les interactions hôte-tumeur et de mettre en évidence de nouveaux biomarqueurs utiles pour le diagnostic précoce et la surveillance de la maladie sous traitement. Le premier objectif de ma thèse consistait à étudier la sécrétion par les cellules malignes d’une famille de microARN viraux appelés miR-BART et leur diffusion dans le sang périphérique chez les sujets porteurs de tumeurs associées à EBV. Pour la première fois j’ai mis en évidence une sécrétion d’exosomes porteurs de miR-BART par les cellules de NPC en culture in vitro. J’ai également montré que les miR-BART, particulièrement miR-BART7, sont détectables dans le plasma de sujets porteurs de NPC. Contrairement à ce qui se passe in vitro les miR-BART plasmatiques ne sont pas transportés par des exosomes. Des données obtenues chez la souris montrent qu’ils peuvent être transportés par des complexes extra-cellulaires que l’on peut précipiter au moyen d’anticorps anti-ago2. Nous cherchons à confirmer ces données sur des échantillons de plasma provenant de patients porteurs de NPC. Ces données pourront guider à l’avenir l’utilisation des miR-BART circulants comme source de biomarqueurs.Le deuxième volet de ma thèse avait pour but d’étudier les modifications du protéome des exosomes induites par une oncoprotéine du virus d’Epstein-Barr appelée LMP1 (latent membrane protein 1). J’ai montré que la LMP1, lorsqu’elle est exprimée dans les cellules lymphocytaires ou épithéliales, infectées ou non par EBV, induit la libération de la protéine PARP1 dans le milieu extra-cellulaire. Cette PARP1 extra-cellulaire n’est pas associée aux exosomes ni aux microvésicules mais à des nano-objets non-vésiculaires contenant notamment des histones et de l’ADN. Nous avons désignés ces objets sous le terme de complexes ADN-protéines extra-cellulaires. Nous ne savons presque rien de la biogenèse de ces complexes ; nous pensons qu’ils ne proviennent pas uniquement de cellules en apoptose. En revanche, des expériences préliminaires suggèrent que la présence de PARP1 dans ces complexes coïncide avec une activation permanente de la PARP1 induite dans les cellules productrices par l’expression de l’oncoprotéine LMP1. Cette hypothèse est en cours de vérification grâce à des expériences menées sur des lignées cellulaires exprimant différentes formes sauvages ou mutées de la LMP1. Ces données sur l’activation de la PARP1 et sur sa sécrétion induite par la LMP1 auront des retombées intéressantes pour notre compréhension des mécanismes d’oncogenèse et d’auto-immunité liés à l’infection par le virus d’Epstein-Barr
The study of tumoral microenvironment should take into account different modes of intercellular communications: direct contacts between extracellular membranes, secretion and uptake of cytokines and finally emission and uptake of complex biological objects like exosomes and microvesicles.Epstein-Barr virus (EBV) is associated with several human malignancies of epithelial origin (Nasopharyngeal carcinoma or NPC) or of lymphoïd origin (post-transplant lymphoproliferative disorder or PTLD). In these tumors, malignant cells are latently infected by EBV and release exosomes and microvesicles containing viral nucleic acids and proteins. Studying them will enable a better understanding of tumor-host interactions and the discovery of new markers which could be useful for early diagnostic and the follow-up of the disease under treatment.The first aim of this thesis was to study the release by malignant cells of EBV microRNAs belonging to the BART family and their blood diffusion in patients bearing NPC tumors. For the first time, I’ve shown that exosomes released by NPC cells in vitro contain EBV miR-BART microRNAs. Moreover, ebv-miR-BART7 can be detected in the plasma of NPC patients. Unlike what is observed in vitro, circulating BART microRNAs are not carried by exosomes. Recent data from studies in xenografted mice show that they are carried by extra-cellular complexes which can be immunoprecipitated by anti-Ago2 antibodies. We are currently trying to confirm these data in plasma from NPC patients. This work will ease the use of miR-BARTs as potential biomarkers.The second aim was to study the proteome modifications induced by the EBV Latent Membrane Protein 1 protein (LMP1). I’ve shown that LMP1 expression in lymphoid or epithelial cells infected or not by EBV induces the release of PARP1 in the extra-cellular space. This extra-cellular PARP1 is not carried by exosomes or microvesicles but is embedded in non-vesicular nano-objects containing histones and DNA. We have called these objects “DNA-proteins complexes”. We don’t know how they are produced and released by cells. We think that they are not only secreted by apoptotic cells. Recent data show that this release of extra-cellular PARP1 is associated with PARP1 activation by LMP1 oncoprotein expression. We are trying to prove this hypothesis using cell lines expressing wild type or mutated LMP1. The release and the activation of PARP1 induced by LMP1 expression will help to understand the mechanisms of EBV-associated oncogenesis and auto-immunity

A significant number of patients requiring adoptive T-cell therapy (ATCT) need to resort to third party donors; we aimed to find ways to optimise ATCT from third party donors in EBV-associated diseases. Firstly, we evaluated the T-cell response to 29 EBV-restricted peptides in a cohort of 100 healthy donors. For each peptide we found at least one high-responding donor. Also, we compared the efficacy of different separation techniques. These results support the setting up of a registry of third party donors, to provide fresh EBV-specific T cells for ATCT. Secondly, we investigated the mechanisms generating T memory stem cells (T_SCM), which are considered most suitable for ATCT. We demonstrated that homeostatic cytokines revert recently differentiated CD8⁺ memory T cells from cord blood (CB) to cells with a T_N-like phenotype (T_Nrev) and T_SCM-like characteristics. Finally, we compared phenotype and function of CD8⁺ T cells from peripheral blood and CB, after transduction of an EBV-specific TCR. Transduction efficiency, growth kinetics and cytolytic activity were comparable. However, TCR-transduced CB T cells showed less differentiated phenotype, increased multi-cytokine expression, and lacked expression of the senescence marker CD57. These data suggest that survival of engineered T cells in vivo is likely to be improved by using cells from CB.

Multiple sclerosis (MS) is a debilitating disease in which the immune system aberrantly targets the central nervous system (CNS). There is compelling evidence that Epstein-Barr virus (EBV) is associated with MS development but the pathogenic mechanisms are unknown. The molecular mimicry hypothesis suggests the immune response to EBV, which normally would restrain the virus infection, mistakenly targets CNS components. This thesis characterised humoural and cellular responses to the virus in healthy controls (HC) and MS patients, increasing the range of EBV and CNS proteins investigated and seeking evidence of crossreactivity as predicted by the hypothesis. Compared to HC, patients had elevated EBNA1 and virus capsid antigen-specific antibody responses. EBNA3-specific antibody responses were also more frequently detected in patients, a previously undescribed observation. Both groups had similar frequencies of circulating Tcells specific for autologous lymphoblastoid cell lines (LCL) or EBNA1, although minor differences in cytokine profile were detected. LCL-specific T-cell cultures established from both patients and HC exhibited cross-reactivity to CNS antigens. This result supports a role for molecular mimicry but also suggests that other unknown or more complex factors must influence MS development. While such T-cells are a necessary prerequisite for the molecular mimicry hypothesis, their presence in HC suggests other factors must influence MS development. Identification of these factors must be a priority for future studies.

Classical Hodgkin lymphoma (cHL) is one of the commonest lymphomas in the developed world, frequently affecting young adults. The majority of patients will be cured of their disease, but the toxicities of the therapy required to achieve this can lead to long-term morbidity in survivors. In addition, whilst most patients are cured, there remains approximately 15% -20% of patients who will not respond to primary therapy and may ultimately die of their disease. Approximately one-third of cases of cHL in the developed world are associated with the Epstein-Barr virus (EBV), where this association is believed to be causal. A ubiquitous herpesvirus, persistently infecting more than 95% of the world’s population, precisely how this virus causes malignant disease in a minority of immune competent hosts remains ill-understood. Epidemiological evidence points to inherited genetic factors. Long-recognised to have an association with the class I human leukocyte antigen (HLA) system, recent studies have confirmed that risk of EBV-associated cHL is related to an individual’s HLA-A* allotype, with HLA-A*01:01 being associated with increased risk of disease and HLA-A*02:01 being protective. Heterozygotes are observed to have an intermediate risk. HLA plays a central role in the recognition and cell killing of virally-infected or malignant cells by the cytotoxic T lymphocytes (CTLs) of the cell-mediated immune system. The exact mechanism whereby HLA-A* exerts its effect on risk of cHL unknown, but CTL responses to EBV in this context are hypothesised to be crucial. The CTL response to EBV is well-studied. Immunodominant epitopes restricted through common class I alleles have been described, many directed towards peptides derived from proteins expressed in the lytic cycle of viral infection. In spite of intensive study, no confirmed HLA-A*01:01-restricted EBV-specific CTL responses have been described, raising the possibility that absent or weak CTL responses specifically to EBV might lead to elevated risk of disease. However, particularly given the intermediate risk of disease seen in HLA-A*01:01 heterozygotes, it remains a possibility the HLA-A*01:01-associated risk might be due to qualitative or inhibitory changes to the EBV-specific immune response. The work of this thesis set out to address a number of specific questions regarding the role of HLA class I in the aetiology and clinical outcome of cHL. Firstly, whether an HLA-A*01:01 allele could modify the magnitude of the CTL response to HLA-A*02:01-restricted epitopes was examined. In a study of healthy adults examining CTL responses using interferon-γ ELISPOT, overall HLA-A phenotype did not significantly affect the EBV-specific CTL response restricted through HLA-A*02:01. However, exploratory analysis of cytokine levels in response to stimulation with EBV peptides demonstrated significantly higher secretion of IL-10 (with a nearly 10-fold difference), IL-17 and IL-5 in response to stimulation with EBV peptides in HLA-A*02:01/A*01:01 heterozygotes, compared to other HLA-A*02:01 phenotype groups. This suggests a possible effect of HLA-A*01:01 in HLA-A*02:01/A*01:01 heterozygotes which might begin to explain some of the HLA-associated differences in risk of developing EBV+ve cHL. Secondly, again in a study of healthy EBV-seropositive adults, and using sensitive methodologies, HLA-A*01:01-restricted EBV-specific CTL responses were sought, and, in an exploratory analysis, cytokine responses were examined. No HLA-A*01:01-restricted CTL responses to EBV were detected in this study, however, exploratory analysis demonstrated statistically significant differences in cytokine levels following simulation with EBV, with HLA-A*01:01 homozygotes generating much higher levels of IL-6. Lastly, given the importance of class I HLA in determining risk of developing EBV+ve cHL, a study of 424 patients with cHL was performed to determine if HLA-A*01:01 and A*02:01 alleles are a factor in determining clinical outcome. In this study, HLA-A*02:01 was associated with inferior overall survival (OS) and disease-specific survival (DSS) in EBV+ve cHL, and was independently prognostic in an adjusted analysis. Given the extremely poor outcomes seen in this study in HLA-A*02:01 carriers with EBV+ve disease (61.7% 10-year OS), it is possible that this group of patients is not currently being well-served by standard first-line therapy and may benefit from novel therapies.

Background. Head and neck cancers (HNC) are among the most common malignancies worldwide, and about 90–92% of oral neoplasias are oral squamous cell carcinomas (OSCC). Alcohol and tobacco consumption have been recognized as the main risk factors for OSCC development. Oncogenic viruses, such as human papillomavirus (HPV) or Epstein-Barr virus (EBV), as well as genetic alterations may also contribute to tumour formation.  Aims. To study the prevalence of HPV, EBV, Herpes simplex type-1 (HSV-1), and HPV-16 and their integration status as well as the molecular mechanisms that can serve as a basis for the development of OSCC. Results. In Paper I we reported a statistically significant increase in the prevalence of HPV-16 in oral epithelial dysplasia (OED) and OSCC samples compared to controls. A statistically significant increase was also seen in integrated HPV-16 compared to episomal viral forms when comparing OED and OSCC samples. Paper II reported the detection of HSV-1 in 54% of healthy samples, in 36% of oral leukoplakia samples, and 52% of OSCC samples. However, these differences were not statistically significant. In Paper III we reported a statistically significant increase in the detection of HPV-positive samples when comparing nested polymerase chain reaction (PCR) with single-PCR results in OSCC and fresh oral mucosa. Paper IV reported that the highest prevalence of HPV (65%) was seen in Sudan, while an HSV-1 prevalence of 55% and an EBV prevalence of 80% were seen in the UK. Finally, Paper V reported that the mRNA levels of Bcl-2, keratin 1, keratin 13, and p53 were significantly lower and that the level of survivin was significantly higher in the OSCC samples of the toombak users than in their paired control samples. Significant downregulation in keratin 1 and keratin 13 expression levels was found in the OSCC samples of the non-toombak users relative to their normal control samples. Conclusion. HPV-16 integration was increased in oral epithelial dysplasia and OSCC compared to normal oral mucosa. Nested PCR is a more accurate method of establishing HPV prevalence in samples containing low copy numbers of HPV DNA. HPV and EBV may be a risk factor in OSCC development. Our findings confirmed the role of survivin in OSCC carcinogenesis and survivin might be interesting as a biomarker to be monitored. The results presented here provide both clinical and biological insights that will bring us closer to the goal of managing this disease and improving treatment and outcomes for future patients.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Cervical cancer is the second largest cause of deaths from malignant cancer among women worldwide. Many studies show the importance of the HPV virus in the onset of cervical cancer, but because of the monoclonal nature of this cancer, it is suspected that HPV infection may not be considered the only causal factor responsible for the development of cervical carcinoma. Studies have indicated that EBV may have a role in cooperation with the HPV16 tumor development. Such cooperation could influence the tumor microenvironment by creating a favorable niche for its progression. Cancer cells secrete enzymes that degrade the extracellular matrix (ECM). Such enzymes such as matrix metalloproteinases (MMPs) facilitate cell movement in tissues and induce the activity of growth factors that promote angiogenesis. Among the group of MMPs, MMP-7 seems to have a crucial role in the development of carcinogenesis, since its action has been associated with the cleavage and release of adhesion molecules important. In this sense, this study was performed to establish the relationship between the HPV coinfection / EBV and expression of MMP-7. They were examined immunohistochemically histological slides of cervical lesions biopsies of 60 patients aged 35 to 65 years. The samples were divided into high grade lesion and low grade and evaluated semiquantitatively by scoring system ranging from 0 to +++. Histopathological indexes the degree of injury were established. It observed a statistically significant association between high-grade lesion, the expression of MMP-7 and the presence of the HPV virus, with p = 0.007 at the junction portion squamocolumnar and p = 0.023 in the portion of the ectocervix. This correlation
O câncer cervical é responsável pela segunda maior causa de mortes por neoplasia malignas entre as mulheres no mundo. Muitos estudos comprovam a importância do vírus HPV no surgimento do câncer cervical, porém devido à natureza monoclonal desta neoplasia, suspeita-se que a infecção pelo HPV não possa ser considerada o único fator causal responsável pelo desenvolvimento do carcinoma de colo de útero. Estudos já apontaram que o EBV pode ter um papel de cooperação com o HPV16 no desenvolvimento tumoral. Tal cooperação poderia influenciar o microambiente tumoral criando um nicho favorável para a sua progressão. Células de câncer secretam enzimas que degradam a matriz extracelular (MEC). Tais enzimas, como as metaloproteinases de matriz (MMPs) facilitam a movimentação celular nos tecidos e induzem a atividade de fatores de crescimento que promovem a angiogênese. Dentre o grupo das MMPs, a MMP-7 parece ter papel crucial no desenvolvimento da carcinogênese, pois sua ação já foi associada com a clivagem e a liberação de importantes moléculas de adesão. Neste sentido, foi realizado este estudo para estabelecer a relação entre a co-infecção HPV/EBV e a expressão da MMP-7. Foram examinadas imunohistoquimicamente lâminas histológicas de biópsias de lesões de colo uterino de 60 pacientes com idades entre 35 e 65 anos. As amostras foram divididas em lesão de alto grau e de baixo grau e avaliados semiquantitativamente através de um sistema de escore variando de 0 à +++. Índices histopatológicos quanto ao grau das lesões foram estabelecidos. Foi observada uma associação estatisticamente significante entre a lesão de alto grau, a expressão da MMP-7 e a presença do vírus HPV, com p=0,007 na porção da junção escamocolunar e p=0,023 na porção da ectocérvice.

Les microtubules (MT) sont des polymères dynamiques ancrés par leurs extrémités moins aux centres de nucléation alors que leurs extrémités plus, explorent le cytoplasme, jusqu’à être stabilisées. Cette capture des extrémités permet l’organisation du réseau des MT. Les +TIP sont un groupe de protéines qui s’associent aux bouts plus des MT. EB1 est une protéine centrale dans le réseau des +TIP qui régule la dynamique des MT et leur interaction avec les structures d’ancrage des extrémités plus. Par protéomique ciblée, nous avons caractérisé l’interactome d’EB1, et mis en évidence un groupe de protéines, précédemment associées aux centres de nucléation incluant AKAP9, une protéine échafaudage pour les protéines kinases A (PKA), la protéine de la matrice péricentriolaire CDK5RAP2, et une isoforme courte de la myomégaline que nous avons appelé SMYLE (Short MYomegalin Like EB1 binding protein). La cartographie moléculaire a permis de montrer que ces protéines formaient un complexe organisé de manière hiérarchique. Nous avons observé que l’association transitoire deLa protéine SMYLE (Short MYomegalin Like EB1 binding protein )dans l'organisation d'un complexe centrosomal, la régulation de la nucléation et la stabilisation des microtubules : conséquences sur la migration et la division des cellules cancéreuses avec les MT néo-nucléés au centrosome favorisait la nucléation et l’acétylation des MT. De manière notable, la déplétion de SMYLE aboutissait à un défaut de nucléation, mais aussi de la capture corticale des MT. Ces défauts dans l’organisation des MT étaient associés à une baisse notable de la migration des cellules de carcinome mammaire et à des anomalies mitotiques. Nos résultats nous permettent de proposer que SMYLE fait partie d’un complexe centrosomale, qui favorise l’assemblage ou la stabilité des microtubules néo-nucléés, contribuant ainsi à des processus majeurs pour le développement tumoral
Microtubules (MT) are dynamic polymers anchored by their minus ends at the MT organizing centers while their highly dynamic plus end explores the cytoplasm until it get stabilized. This plus end capture allows the organization of the MT network. +TIPs are a group of proteins that share the commonality to associate either directly or indirectly to MT plus ends. EB1 is a central protein of the +TIP network that regulates MT dynamics and their interactions with plus end anchoring structures. Using targeted proteomics, we have characterized the EB1 interactome and revealed a set of protein previously shown to associate with the nucleating centers that included AKAP9 an anchoring protein for protein kinase A (PKA), the pericentriolar matrix protein CDK5RAP2 and a short Myomegalin isoform that we named SMYLE (Short MYomegalin Like EB1 binding protein). Molecular mapping revealed that the proteins formed a hierarchically organized complex. We have observed that the transient association of SMYLE to the newly nucleated MTs at the centrosome favored the nucleation and acetylation. Interestingly, SMYLE depletion led to MT nucleation defects, but also a disruption of cortical MT capture. These defects in the MT network were associated with a steep fall in the migratory potential of breast cancer cells and mitotic abnormalities. Our results allow proposing that SMYLE belongs to centrosomal supramolecular complex that favors the assembly and stability of newly nucleated MTs, thus contributing to major processes in tumor development

Orientadores: Ubirajara Ferreira, Valéria Cagnon Quitete
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O tratamento do câncer de bexiga urotelial não músculo invasivo (CBNMI) com Bacilo Calmette-Guerin (BCG) tem efeito comprovado na redução de recidiva tumoral, embora ocorram efeitos colaterais de intensidades variadas, desde sintomas irritativos leves até reação sistêmica grave e o impacto na progressão tumoral seja controverso. Neste cenário a enterotoxina B do Staphylococcus aureus (EBS) destaca-se como alternativa promissora na terapêutica do CBNMI. Assim, os objetivos principais do presente estudo foram comparar e caracterizar os efeitos morfológicos, antiangiogênicos e o balanço entre proliferação e apoptose das imunoterapias com BCG, EBS e BCG-EBS no tratamento do CBNMI de ratos induzidos quimicamente. Um total de 75 ratas da linhagem Fisher 344 foram utilizadas. Para a indução do câncer, 60 animais foram anestesiados e receberam uma dose intravesical de 1,5mg/kg de N-metil-N-nitrosouréia (MNU) a cada 15 dias, nas semanas 0, 2, 4 e 6 (4 doses). Os outros 15 animais receberam 0,3ml de solução fisiológica intravesical e constituíram o grupo Controle (CT). Após o período de indução com MNU, os animais foram divididos em 5 grupos (15 animais cada): grupo MNU: recebeu o mesmo tratamento que o grupo CT - 0,3 ml de solução fisiológica intravesical; grupo BCG: recebeu dose intravesical de 106 UFC (2x106 UFC/mg) de BCG; grupo EBS: recebeu dose intravesical de 10?g/ml de EBS; grupo BCG-EBS: recebeu tratamento simultâneo com BCG e EBS nas mesmas concentrações que nos grupos BCG e EBS; todos por 6 semanas consecutivas. Após 15 semanas de protocolo, os animais foram sacrificados e as bexigas urinárias coletadas e submetidas às análises histopatológica, imunohistoquímica e Western Blotting. Os resultados demonstraram que as alterações histopatológicas mais frequentes em cada grupo foram carcinoma papilífero e carcinoma in situ no grupo MNU, hiperplasia papilífera nos grupos BCG e EBS e hiperplasia plana no grupo BCG-EBS. Não houve toxicidade relevante. Os índices apoptótico e proliferativo foram aumentados em todos os grupos de tratamento. Contudo, esses índices foram menores nos grupos BCG, EBS e BCG-EBS em relação ao grupo MNU. Intensas imunorreatividades para VEGF, MMP-9 e IGFR-1 foram verificadas no grupo MNU; moderadas nos grupos BCG e EBS; e fracas nos grupos BCG-EBS e Controle. Em contraste, intensa imunorreatividade para endostatina foi verificada nos grupos Controle e BCG-EBS em relação aos demais grupos experimentais. Assim, pode-se concluir que o tratamento associado BCG-EBS foi mais eficaz em restaurar o equilíbrio entre os processos de apoptose e proliferação celular e as alterações histopatológicas do que os tratamentos isolados com BCG e EBS. Além disso, a diminuição da proliferação celular e das alterações histopatológicas correlacionaram-se com o aumento da reatividade para endostatina e diminuição das reatividades para VEGF, IGFR-1 e MMP-9. A associação entre BCG e EBS abre uma nova perspectiva terapêutica no CBNMI
Abstract: PURPOSE: Compare and characterize effects of intravesical Bacillus Calmette-Guerin (BCG), Staphylococcal Enterotoxin B (SEB) and BCG plus SEB treatments in the non-muscle invasive bladder cancer (NMIBC). METHODS: Seventy-five female Fisher 344 rats were anesthetized and 15 of them received 0.3 ml of saline (Control group), 60 animals received 1.5 mg/kg dose of N-methyl-N-nitrosourea (MNU), all intravesically every other week for 6 weeks. The rats were divided into 5 groups: MNU and Control groups received 0.3 ml of saline; BCG group received 106 CFU dose of BCG; SEB group received 10 ?g/ml dose of SEB and BCG-SEB group received simultaneous BCG and SEB, all intravesically for 6 weeks. After 15 weeks, all urinary bladders were collected for histopathological, immunological and Western Blotting analyses. RESULTS: Papillary carcinoma (pTa) and high-grade intraepithelial neoplasia (carcinoma in situ) were more often in MNU group, papillary hyperplasia in BCG and SEB groups and flat hyperplasia in BCG-SEB group. No significant toxicity was observed. Apoptosis and cellular proliferation indexes decreased in BCG, SEB and BCG-SEB groups compared to MNU group. Intensified VEGF, MMP-9, Ki-67 and IGFR-1 immunoreactivities were verified in the MNU group; moderate in the BCG and SEB groups; and weak in the BCG-SEB and Control groups. In contrast, intense endostatin immunoreactivity was verified in the Control and BCG-SEB groups. CONCLUSIONS: BCG and SEB presented similar anti-angiogenic effects. BCG-SEB treatment showed an additional activity compared to monotherapy, being more effective in restoring apoptosis and cellular proliferation balance, correlating with increased endostatin and decreased VEGF, MMP-9, Ki-67 and IGFR-1 reactivities
Doutorado
Fisiopatologia Cirúrgica
Doutor em Ciências

Angesichts der nach wie vor hohen Mortalität und Morbidität des Bronchialkarzinoms ist die Entwicklung geeigneter Methoden zur früheren Diagnostik eine wichtige Notwendigkeit, um die geringe durchschnittliche 5-Jahres-Überlebensrate von 15% – 18% zu steigern. Unter diesem Gesichtspunkt wurde in der vorliegenden Arbeit das Atemkondensat von Patienten mit Bronchialkarzinom als nicht-invasiv und kostengünstig zu gewinnendes Medium auf das Vorliegen eines potentiellen Screeningmarkers – dem methylierten Tumorsuppressor-Gen p16 – untersucht. Dazu wurde ein Versuchsablauf entwickelt, bei dem trotz des geringen DNA-Gehaltes im Atemkondensat p16-Methylierungen nachgewiesen werden konnten. Die letztendlich etablierte Methode war eine methylierungsspezifische nested-PCR mit anschließendem Restriktionsverdau durch das Restriktionsenzym BstUI. Des Weiteren erfolgte die Untersuchung von in Paraffin eingebetteten Tumorpräparaten der Patienten. In der anschließenden statistischen Auswertung wurde der Einfluss von verschiedenen Faktoren wie COPD-Grad, Tumorlage, Tumorart, Nikotinabusus und stattgehabte Chemo- oder Strahlentherapie auf den Methylierungsstatus des p16-Gens analysiert.

Le cancer papillaire de la thyroïde (CPT) est la tumeur endocrine la plus fréquente. Des anomalies moléculaires activant la voie des MAPK (Mitogen-Activated Protein Kinases) sont identifiées, de façon mutuellement exclusive, dans environ 70% des cas. Il s’agit de réarrangements chromosomiques, le plus souvent de type RET/PTC (10%), de mutations ponctuelles activatrices des trois isoformes de l’oncogène RAS (H, N et K-RAS) (10%), ou de l’oncogène B-RAF (50%). La mutation « hot spot » B-RAFV600E est la plus fréquemment identifiée, elle est associée à une plus grande agressivité clinique (diagnostic à un stade tardif, risque de récidives et de décès accru). Ces évènements moléculaires ont pour conséquence commune l’activation de la voie des MAPK, se traduisant en aval par la phosphorylation de MEK (Mitogen-activated Extracellular signal-Regulated Kinase) puis de ERK (Extracellular signal-Regulated Kinase). Cette dernière est régulée négativement par des phosphatases, appartenant à la famille des Dual Specificity Phosphatases (DUSPs), d’expression ubiquitaire, et en particulier de deux phosphatases spécifiques de ERK, l’une cytoplasmique (DUSP6) et l’autre nucléaire (DUSP5). Nous avons fait l’hypothèse que ces phosphatases pouvaient être soit des gènes suppresseurs de tumeurs (leur perte d’expression conduisant à une augmentation de phosphorylation de ERK et une prolifération accrue), soit des marqueurs du degré d’activation de la voie MAPK dans le cadre d’une boucle de rétrocontrôle négatif. Ceci nous a conduits à analyser la régulation et l’expression de ces phosphatases dans trois modèles : la lignée cellulaire PCCL3 (thyroïde de rat), exprimant l’un des trois principaux oncogènes mutés dans les CPT (RET/PTC3 ou H-RASV12 ou B-RAFV600E) sous le contrôle d’un promoteur inductible par la doxycycline, des lignées cellulaires humaines dérivant de CPT et des CPT humains. (...)
Papillary thyroid cancer (PTC) is the most common endocrine malignancy. Mutually exclusive and activating alterations of the MAPK pathway (Mitogen-Activated Protein Kinases) are identified in 70% of cases. Common mutations found in PTCs are point mutation of the B-RAF (50%) and RAS genes (10%) as well as RET/PTC chromosomal rearrangements (10%). The hot spot B-RAFV600E mutation is the most frequently alteration identified and is connected with agressive clinical characteristics (high stage at diagnosis, high recurrence risk and death). These molecular events lead to constitutive activation of the MAPK pathway, resulting in MEK (Mitogen-activated Extracellular signal-Regulated Kinase) and ERK (Extracellular signal-Regulated Kinase) phosphorylation. ERK is negatively regulated by phosphatases and among them, Dual Specificity Phosphatases (DUSPs), ubiquitary expressed, in particular two ERK-specific phosphatases DUSP5 (nuclear) and DUSP6 (cytosolic). We hypothesized that these phosphatases could have tumor supressor properties (i.e. their loss would be associated with an increase in MAPK pathway activation) or may serve as a surrogate marker of MAPK pathway activation in the context of a negative feedback loop. We analysed regulation and expression of both phosphatases in 3 models: three PCCL3 cell lines (rat thyroid cells) expressing one of the most common oncogene identified in PTCs (RET/PTC3 or H-RASV12 or B-RAFV600E) under the control of a doxycycline-inducible promoter, human PTC-derived cell lines and human PTC. We demonstrated that MAPK pathway activation was correlated with induction of DUSP5 and DUSP6. These phosphatases are involved in a negative feedback loop that contributes to a tight regulation of phospho-ERK levels. DUSP5 and DUSP6 mRNA are overexpressed in human PTCs, especially in B-RAF mutated tumors suggesting a higher MAPK signaling output in these agressive PTCs. Silencing of DUSP5 and/or DUSP6 by small interfering RNA does not affect proliferation of human B-RAFV600E thyroid carcinoma-derived cell lines, suggesting the lack of tumor suppressor gene role. Compensatory changes in expression of DUSPs when a specific one is inactivated may explain this lack of effect. On the opposite, a DUSP6 pharmacological inhibitor induced a concentration dependent decrease in proliferation of human B-RAFV600E cells, suggesting « off-target » effect of this inhibitor. In a second part, we analysed the regulation of DUSP5 expression, which is a target of the MAPK pathway activation. We demonstrated, using pharmacological inhibitors, that DUSP5 is an early response gene, regulated mostly by the MAPK pathway, at the transcriptional level. Two contiguous CArG boxes that bind serum response factor (SRF) were found in a 1Kb promoter region, as well as several E twenty-six transcription factor family binding sites (EBS). These sites potentially bind Elk-1, a transcription factor activated by ERK1/2. Using wild type or mutated DUSP5 promoter reporters, we demonstrated that SRF plays a crucial role in serum induction of DUSP5 promoter activity, the proximal CArG box being important for SRF binding in vitro and in living cells. Moreover Elk-1 was bound in vitro to a promoter region containing the proximal CArG box and a putative EBS. Its specific binding to SRF was necessary to elicit promoter response to dominant positive Elk-VP16 and to enhance the response to serum stimulation. Altogether our results suggest that the MAPK pathway is more active in B-RAFV600E PTC than in PTC with other genetic alteration and could explain their clinical agressivity. DUSP5 and DUSP6, as well as phosphorylated MEK, are markers of activation of the MAPK pathway. Neither phosphatase has tumor suppressor properties in our thyroid cancer cell models. Our results suggest redundancy and functional compensation among DUSPs. (...)

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line that can undergo spontaneous differentiation when grown past confluency, we observed a loss of VCAM1 (vascular cell adhesion molecule-1) expression while ICAM1 (intercellular cell adhesion molecule-1) expression was seen to be stable in the course of differentiation. Protein kinase C theta (PKC&theta
) acted downstream of PKC to inactivate Inhibitor of kappa B (IB) and activate NF-&kappa
B in the undifferentiated cells and this axis was inhibited in the differentiated cells. The increase in ICAM1 expression in the differentiated cells was due to a transcriptional upregulation by C/EBP. The protein expressions of both ICAM-1 and VCAM-1, however, were found to decrease in the course of differentiation, with both proteins getting post-translationally degraded in the lysosome. Functionally, a decrease in adhesion to HUVEC cells was observed in the differentiated Caco-2 cells. Thus, the regulation of ICAM-1 and VCAM-1, although both NF-B target genes, appear to be different in the course of epithelial differentiation. microRNAs are known to regulate many cellular pathways. miR-146a, which is known to target NF-&kappa
B, was shown to be highly upregulated in differentiated Caco-2 cells. As a predicted target of miR-146a, mRNA and protein expression of MMP16 was inversely correlated with miR-146a during differentiation of Caco-2 cells. miR-146a could bind to the 3&rsquo
UTR of MMP16 and ectopic expression of miR-146a resulted in a decreased mRNA and protein expression of MMP16 in the undifferentiated Caco-2 and HT-29 cells. Functionally, decreased gelatinase activity determined by gelatin zymography and reduced invasion and migration through Transwells was observed. In the final part of the thesis, the inhibition of NF-&kappa
B via PPAR&gamma
in 15-Lipoxygenase-1 (15LOX1) expressing cells was investigated. The expression of 15LOX1, a member of the inflammatory arachidonate cascade, could lower phosphorylation of I&kappa
B&alpha
and NF-&kappa
B DNA binding activity which was reversed with a 15LOX1 inhibitor. This inhibition was mediated by phospho-PPAR&gamma
, which in turn was phosphorylated by ERK1/2.

Le traitement du cancer par thérapie génique nécessite d’une part des gènes suicides efficaces et, d’autre part, l’adressage spécifique de ces gènes aux cellules cancéreuses. J'ai d'abord caractérisé un nouveau gène suicide dérivé de la désoxycytidine kinase humaine (dCK) : le M36. Comparé à la dCK, le M36 permet une meilleure sensibilisation des certaines cellules cancéreuses aux traitements avec différents chimiothérapeutiques comme la gemcitabine et la cytarabine. Ces résultats sont particulièrement encourageants pour l'élimination des cellules cancéreuses résistantes à ces traitements du fait d’un défaut de la dCK. Dans une deuxième partie, je me suis intéressée à l'adressage spécifique des transgènes aux cellules cancéreuses par les vecteurs lentiviraux. J'ai travaillé à la preuve de concept qu’une enveloppe (Env) VIH modifiée peut permettre un tel ciblage. J'ai généré une Env qui a fortement diminué son tropisme naturel et qui comporte un motif liant le marqueur tumoral modèle HER2
Cancer gene therapy requires the use of an effective suicide gene and the specific targeting of cancer cells. In my PhD work, I have first characterized a new potential suicide gene derived from human deoxycytidine kinase (dCK): M36. Compared to dCK, M36 improves sensitization of certain cancer cells to treatment with chemotherapeutic compounds as gemcitabine and AraC. These results are particularly encouraging for the elimination of cancer cells resistant to the treatment because of a defect with dCK. In a second part, I have worked at the proof of concept that a modified HIV envelope can allow specific targeting of cancer cells by lentiviral vectors. During this work, I have generated a CD4i envelope with a strongly diminished natural tropism and that carries a motif known to bind the model cell surface cancer marker HER2. This envelope constitutes a good starting material to be improved by evolution in cell culture to obtain specific targeting of HER2+ cells

Le récepteur nucléaire Fetoprotein Transcription Factor (FTF) identifié par notre laboratoire et exprimé principalement dans le système digestif est un régulateur important du métabolisme des lipides et des stéroïdes, de la prolifération cellulaire et du développement embryonnaire. Plusieurs groupes ont constaté que l’influence du récepteur FTF sur la synthèse de stéroïdes et la régulation du cycle cellulaire stimule la prolifération tumorale de cellules d’origine tissulaire diverse. Mes études de doctorat ont porté sur l’expression tissulaire de FTF, sur la caractérisation d’un nouvel élément régulateur de son promoteur et sur l’identification par immunoprécipitation de chromatine (ChIP-chip) des cibles transcriptionnelles de FTF dans le foie de souris fœtale et adulte et dans les cellules d’hépatome humain. Ces études ont permis de mieux définir le rôle métabolique de FTF ainsi que son rôle développemental et son implication potentielle dans la carcinogenèse hépatique. L’expression de FTF par les organes du système digestif et par certaines structures nerveuses, sa régulation par des récepteurs nucléaires métaboliques et sa liaison aux promoteurs de multiples enzymes et transporteurs impliqués dans le métabolisme énergétique placent FTF dans une position clé dans l’homéostasie métabolique et énergétique de l’organisme. Le facteur de transcription C/EBPpartenaire de FTF au promoteur de l’AFP et impliqué lui aussi dans le développement hépatique et le métabolisme énergétique, est lié au promoteur de 20% des cibles transcriptionnelles de FTF. De plus, C/EBP lie le promoteur de FTF formant ainsi une autre boucle activatrice s’ajoutant au réseau transcriptionnel hépatique. Dans les cellules d’hépatome, FTF lie les promoteurs de plusieurs gènes impliqués dans la prolifération et le maintien des cellules tumorales, soit des régulateurs de la réplication, de la croissance et de l’apoptose cellulaire. FTF fait donc partie intégrante du réseau transcriptionnel hépatique régissant le développement et la différenciation hépatique et le maintien du métabolisme énergétique chez l’adulte et est vraisemblablement impliqué dans la promotion de la cancérogenèse hépatique.
FTF is a nuclear receptor principally expressed in adult digestive organs that has been shown to act as a major regulator of lipids and steroids metabolism, cellular proliferation and embryonic development. FTF involvement in steroid synthesis and cell cycle regulation tends toward the stimulation of tumor proliferation in neoplasic tissues in which FTF is expressed. However, more studies of FTF function in normal and disease states and on its regulation are needed to draw a complete picture of FTF activity in cell physiology. Within the context of my studies, I delineated the FTF adult and fetal tissular expression, characterized a novel Ftf promoter element and identified FTF direct hepatic transcriptional targets in fetal, adult and tumor cell lines by using chromatin immunoprecipitation (ChIP-on-chip). These studies defined new FTF functions in metabolism, fetal development and hepatic carcinogenesis. FTF expression in digestive system and in neural structures controlling eating behavior, its transcriptional regulation by metabolic nuclear receptors and its binding to enzyme and transporter gene promoters driving energy metabolism, puts FTF in a key location for governing cellular and organismal energy metabolism. C/EBP, a transcriptional FTF partner on the Afp gene promoter and also involved in energy metabolism, is bound to 20% of the FTF targets including FTF itself thus adding branches to the complex hepatic transcriptional network. In hepatoma cells, FTF binds to proliferation and tumor cell maintenance genes like replication, growth and apoptosis regulators. Therefore, FTF belongs to the hepatic transcription network that governs hepatic development, differentiation and adult energy metabolism and is likely to be involved in promoting hepatic tumorogenesis.

The exponential growth in medical pharmaceuticals and related clinical trials have created a need to better understand the decision-making factors in the processes for developing hospital medication formularies. The purpose of the study was to identify, rank, and compare major factors impacting hospital formulary decision-making among three prescriber groups serving on a hospital's pharmacy and therapeutics (P&T) committee. Prescribers were selected from the University of Texas, MD Anderson Cancer Center which is a large, multi-facility, academic oncology hospital. Specifically, the prescriber groups studied were comprised of physicians, midlevel providers, and pharmacists. A self-administered online survey was disseminated to participants. Seven major hospital formulary decision-making factors were identified in the scientific literature. Study participants were asked to respond to questions about each of the hospital formulary decision-making factors and to rank the various formulary decision-making factors from the factor deemed most important to the factor deemed least important. There are five major conclusions drawn from the study including three similarities and two significant differences among the prescriber groups and factors. Similarities include: (1) the factor "pharmacy staff's evaluation of medical evidence including formulary recommendations" was ranked highest for all three prescriber groups; (2) "evaluation of medications by expert physicians" was ranked second for physicians and midlevel providers while pharmacists ranked it third; and (3) the factor, "financial impact of the treatment to the patient" was fifth in terms of hospital formulary decision-making statement and ranking by all three prescriber groups. Two significant differences include: (1) for the hospital-formulary decision making statement, "I consider the number of patients affected by adding, removing, or modifying a drug on the formulary when making hospital medication formulary decisions," midlevel providers considered this factor of significantly greater importance than did physicians; and (2) for the ranked hospital formulary decision-making factor, "financial impact of treatment to the institution," pharmacists ranked this factor significantly higher than did physicians. This study contributes to a greater understanding of the three prescriber groups serving on a P&T committee. Also, the study contributes to the body of literature regarding decision-making processes in medicine and specifically factors impacting hospital formulary decision-making. Furthermore, this study has the potential to impact the operational guidelines for the P&T committee at the University of Texas, MD Anderson Cancer Center as well as other hospitals.

A nível mundial, o cancro gástrico (CG) é o sexto mais comum com cerca de 1 milhão de novos casos estimados em 2012. Em Portugal, os dados revelam que o CG é o quinto cancro mais frequente com cerca de 3000 novos casos por ano. Para além disso, o CG tem uma elevada taxa de mortalidade sendo responsável por 1387 mortes nos homens e 898 nas mulheres. Dados recentes têm mostrado que o vírus Epstein-Barr (EBV) é detectado em diferentes subtipos histopatológicos de carcinoma gástrico sendo estes tumores responsáveis por aproximadamente 10% de todos os casos. O presente estudo pretende caracterizar os carcinomas gástricos associados ao EBV (EBVaGC) através da sua detecção em tecidos tumorais gástricos de 136 pacientes atendidos no Instituto Português de Oncologia do Porto (IPO Porto FG EPE) no ano de 2011. A detecção de EBV foi realizada por hibridização in situ (ISH) utilizando uma sonda de ADN complementar para ARNs codificados pelo EBV (EBERs). Os resultados demonstraram que os carcinomas associados ao EBV representam 6,6% de todos os casos de CG. Analisando a distribuição de EBV entre os diferentes tipos histológicos, observou-se que o EBV estava presente em 6,6% dos tipos intestinais, em 11,1% dos tipos indeterminados e em 100% dos linfoepiteliomas, contudo nenhum caso foi detectado nos carcinomas difusos (p<0,001). A análise de risco, apesar de não ter sido estatisticamente significativa, sugeriu que os pacientes com carcinomas do tipo intestinal (p=0,350; OR=1,98; 95% IC=0,37-10,5) ou indeterminado (p=0,238; OR=2,78; 95% IC=0,55-15,5) apresentam um risco aumentado de ter EBVaGC; enquanto os pacientes com carcinomas difusos (p=0,078; OR=0,14; 95% CI=0,01-2,58) apresentam um risco diminuído de ter EBVaGC. Quanto à localização do tumor, verificou-se que os carcinomas das regiões superiores do estômago apresentam um risco aumentado de ter um EBVaGC (p=0,032; OR=4,68; 95% IC=1,11-19,7). Em conclusão, a infecção por EBV nos carcinomas gástricos em Portugal é semelhante à de outros países. Por outro lado, as características clínico-patológicas apresentaram diferenças quando comparadas com estudos anteriores, principalmente a ausência de EBV nos carcinomas difusos. Este é o primeiro estudo de caracterização dos EBVaGC em Portugal que reforça a necessidade de mais estudos para esclarecer o papel do EBV como biomarcador preditivo/prognóstico no desenvolvimento do cancro gástrico.
Worldwide, Gastric Cancer (GC) is the sixth most common malignancy with nearly 1 million new cases estimated in 2012. In Portugal, data reveal that GC is the fifth most frequent cancer with about 3000 new cases per year. Moreover GC has still a higher mortality rates being responsible for 1387 deaths in men and 898 in women. Recent data showed that Epstein-Barr virus (EBV) has been detected in different histopathological subtypes of gastric carcinoma and EBV-associated gastric carcinoma (EBVaGC) accounts about 10% of all cases. This study pretends to characterize EBVaGC in our population through detection of EBV in gastric carcinoma tissues from 136 consecutive patients attended at Portuguese Institute of Oncology of Porto (IPO Porto FG EPE) in the year of 2011. EBV detection was performed by in situ hybridization (ISH) targeting EBV-encoded small RNA (EBER-ISH) with an EBER-DNA probe. The results showed that in our population EBVaGC represent 6.6% of all GC cases. Analyzing the distribution of EBV among the different histological types, we observed that EBV was present in 6.6% of intestinal-types, 11.1% of indeterminate types, 100% of lymphoepithelioma-like carcinomas and there were no positive cases among diffuse types (p<0.001). The risk analysis revealed that, despite there are not statistically significant differences, patients with intestinal (p=0.350; OR= 1.98, 95% CI=0.37-10.5) and indeterminate (p=0.238; OR=2.78, 95% CI=0.55-15.5) GC have an increased risk of having an EBVaGC; while diffuse GC (p=0.078; OR=0.14, 95% CI=0.01-2.58) have a decreased risk of having an EBVaGC. Regarding tumor location, the results demonstrated that patients with tumors in upper regions of stomach have an increased risk to have an EBVaGC (p=0.032; OR=4.68, 95% CI=1.11-19.7). In conclusion, the EBV infection rate among gastric carcinomas in Portugal is similar to that ascertained in other countries. Conversely, the clinicopathological features showed differences when compared with previous studies, mainly the absence of EBV in diffuse-type gastric carcinomas. This is the first study to characterize EBVaGC in Portugal which reinforce the need of further studies to clarify the role of EBV and to explore its potential value as predictive/prognostic biomarker in gastric cancer development.

Tong Hung Man Joanna.
"June 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 137-149).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

博士
國立臺灣大學
生物化學暨分子生物學研究所
95
Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus estimated to have infected about 95% of the adult population worldwide. EBV is associated with certain malignancies, such as Burkitt lymphoma, Hodgkin lymphoma and nasopharyngeal carcinoma. Several laboratories have reported recently that EBV may also be involved in the pathogenesis of breast carcinoma, the most frequently diagnosed malignancies in women. In our laboratory, the presence of the EBV genome in breast tumors (66/140, 47.14%) and in normal tissues adjacent to breasr tumors (1/12, 8.3%) indicated that EBV was mainly detected in tumors. After statistical data analysis, EBV was correlated with poor prognosis in breast cancer. It suggested that EBV might be related to the development of some breast carcinomas. To investigate the potential role of EBV in breast cancer, we established EBV-infected breast cancer cell lines, MCF7-A and BT474-A. Three EBV latency genes (EBNA1, EBER1, and BARF0) were expressed in EBV-infected breast cancer cells and this pattern was typical of EBV latency I. EBV-infected MCF7-A and BT474-A cells exhibited increased anchorage-independent growth in soft agar. HER2 (also known as Neu, ErbB2) is a member of the epidermial growth factor receptor (EGFR; HER1/ErbB1) family of receptor tyrosine kinases (RTKs), which also includes HER3(ErbB3) and HER4(ErbB4). Amplification and overexpression of the HER2 proto-oncogene is observed in 20–30% of human breast cancers and is correlated with poor prognosis and decreased patient survival. Moreover, EBV infection enhances expression of HER2 and HER3 gene, and formation of HER2/HER3 heterodimer. HER2/HER3 heterodimer also stimulated phosphorylation of the phosphatidylinositol 3''-kinase (PI3K)-dependent effector kinase Akt, as well as that of the MEK-dependent mitogen-activated kinase (MAPK), extracellular signal-regulated kinase (ERK). To realize the role of HER2/HER3 in anchorage-independent growth, cells were seeded in the soft agar in the presence of an anti-HER2 antibody, herceptin, PI3K inhibitor LY294002 or the MEK inhibitor U0126. The ability of colony formation in soft agar was substantially reduced by either herceptin or the PI3K inhibitor LY294002 or the MEK inhibitor U0126 in MCF7-A cells. In the other hand, the oncogenic potential of Wnt proteins in the mammary gland has been amply demonstrated in mouse models, and it is clear that at least distal components of the Wnt/β-catenin pathway are activated in majority of breast carcinomas. The evidence for this includes elevated levels of cytoplasmic β-catenin, the frequent presence of β-catenin in the nucleus, and expression of transcriptional targets of β-catenin such as cyclin D1. In our study, EBV infection enhances expression and accumulation of β-catenin, and activates expression of β-catenin target genes, c-MYC and cyclin D1. Next, it was examined which of the three EBV genes expressed in these cells (EBNA1, EBER1 and BARF0) was responsible for HER2/HER3 and β-catenin expression. Consequently, we showed that BARF0 alone was sufficient to efficiently up-regulate HER2/HER3 and β-catenin expression and promoted tumorigenic activity in MCF7 and BT474 cells by both overexpression and small interfering RNA knock-down. According to the luciferase reporter assay, expression of BARF0 resulted in 9.1- and 8.3-fold transcriptional activation of the HER2 and HER3 promoter construct in MCF7 cells, respectively. Stably BARF0-tranfected MCF7 cells formed more colonies in soft agar than parental cells. Meanwhile, we found that BARF0 enhances the expression of MMP2 in BT474 cells. And then BARF0 promotes BT474 cells migration and invasion ability through activating β-catenin signaling by PI3K/AKT pathway. These findings imply that BARF0 is likely to enhance the breast cancer tumorigenic activity through activating HER2/HER3 and β-catenin signaling pathway.

碩士
國立臺灣大學
生物化學暨分子生物學研究所
91
Breast cancer is the most common fatal malignancy cancer of females in developed countries. From 1970 to 1990, breast cancer is an important cancer of women in Taiwan and the incidence of age in breast cancer trends to early onset. The etiology of breast cancer involves a complex interplay of genetic, hormonal, and dietary factor that are superimposed on the physiological status of the host. At the molecular level, several oncogenes and tumor suppressor genes were shown to be involved in the development of breast cancer. At 1995, EBV was first detected in human breast cancers then several reports further indicated the presence of EBV in the 20%-50% breast tissue. So our laboratory is interested in developing effective analysis to examine the existence of Epstein-Barr virus（EBV）genome and gene product(s) in breast cancer specimens from Taiwan. We want to know what is the role of EBV in breast cancer carcinogenesis. Some reports indicate that one of the EBNA-1 associated cellular protein ,p32/TAP, was important for RNA splicing. Therefore, we propose a hypothesis that through EBNA-1 and p32/TAP interaction, EBV may enhance the relaxation of RNA fidelity of the host cell. Then the consequent result may cause the mutation of TSG101 and ERα (estrogen receptor alpha). In order to test this hypothesis, we compare the mutation frequency of TSG101 and ERα between parental and EBV-infected breast cancer cell lines and some other cancer cell lines. We also want to know wheather EBV will affect the cellular gene(s) in DNA level .Therefore, the status of p53 between parental and EBV-infected cells has been analyzed as well. Our results pointed out that increase of TSG101 aberrant splicing transcripts has been found in EBV-infected HBL-100-A,T47D-A and A431-A cells. In addition, our result indicated that increase of ERα alternative splicing transcripts has been found in these EBV-infected cells. These data strongly support the hypothesis that EBNA-1 and p32/TAP interaction, EBV may enhance the relaxation of RNA fidelity of the host cell. Then the consequent result may cause the mutation of TSG101 and ERα. According to this excited discovery, the inter-relationship of EBV and those genetic alterations will provide important clues to elucidate the possible role of EBV in carcinogensis although the p53 status has not been affected after EBV infection. Our results pointed out that EBV might involve in breast cancer progression. However, many other mechanistic aspects underlying EBV-mediated transcription are still unclear. For instance, how EBV promotes cellular proliferation through TSG101 and ERα? What specific regulatory genes are involved in this growth pathway? What EBV-stimulated cell cycle signal transduction pathways are important in breast carcinogenesis? Are some of the questions remain to be addressed ?

研究背景及目的：EB病毒(EBV)相關性胃癌的發病率約占胃癌的10%。近年來，越來越多的研究表明， EBV相關性胃癌的腫瘤抑制基因發生異常甲基化。然而，EBV的感染對全基因組DNA甲基化的影響尚不清楚。本研究通過分析EBV感染的細胞中全基因組DNA甲基化的情況，篩選出因EBV感染而發生甲基化的基因，並闡明靶基因在胃癌發生過程的作用。
方法：本研究應用穩定轉染EBV的胃癌細胞AGS (AGS-EBV)和無EBV轉染的AGS細胞為模型。採用高解析度的甲基化DNA免疫共沉澱晶片技術(MeDIP-chip)比較AGS-EBV 和AGS全基因組DNA甲基化的變化，並根據基因本體論(GO)，對EBV誘導的甲基化基因進行分類。採用RT-PCR，去甲基化處理及亞硫酸氫鈉測序(BGS)等方法驗證EBV誘導的甲基化基因。同時，採用基因敲除和過表達方法體外研究靶基因的生物學功能：通過細胞活力實驗和集落形成實驗判斷靶基因對細胞增殖的影響；通过流式细胞技术、伤口愈合实验及侵袭实验研究筛选到的靶基因生长抑素受体1（SSTR1）的功能；此外，还通過腫瘤通路基因PCR晶片分析靶基因調控的下游腫瘤相關基因。
结果：EBV編碼的小RNA(EBER)原位雜交方法和EBV潛伏期膜蛋白(LMP2A)的表達均證實AGS-EBV細胞中確實存在EBV的感染。和AGS細胞相比，發現AGS-EBV細胞中DNA甲基轉移酶3b(DNMT3b)的表達和活性顯著增加。AGS細胞中， LMP2A過表達後，DNMT3b的表達和活性也顯著增加。通過MeDIP-chip篩選出AGS-EBV中1,065甲基化有差異的基因，其中886基因為高甲基化。GO分析結果表明這些高甲基化基因參與KEGG信號通路。其中，六個新的高甲基化基因(MDGA2, IL15RA, SCARF2, EPHB6, SSTR1 和 REC8)在AGS-EBV細胞中的表達低於AGS；經過去甲基化處理之後，這些基因的表達水準有顯著增加。
通過深入研究生長抑素受體1(SSTR1)的生物學功能，發現：敲除SSTR1 能促進胃癌細胞的增殖和集落形成；通過调节G1/S期的調節因子，加快細胞進入S期，顯著增加S期的細胞數目。此外，胰腺癌細胞PANC1細胞中，SSTR1的過表達，也進一步證實SSTR1確實是一種腫瘤抑制基因。腫瘤通路基因PCR晶片結果顯示SSTR1通過促進細胞週期抑制因數(包括p15，p16，p21和p27)的表達，同時抑制CDC25A 和Myc的表達，發揮抑制增殖作用，導致細胞週期停滯在G1期，減少細胞增殖。SSTR1也通過減少凋亡相關基因的表達參與細胞凋亡的過程。此外，SSTR1還能顯著下調遷移相關基因的表達。這些結果表明，在EB病毒相關胃癌的發生過程中，SSTR1通過調節細胞週期、凋亡及遷移的有關基因，進而抑制細胞增殖，減少細胞的遷移和轉移。
结论：AGS感染EBV後，通過LMP2A促進DNMT3b的表達，激活DNMT3b的活性，導致886個腫瘤相關基因发生甲基化。SSTR1是一種EBV誘導的新的甲基化基因，在胃癌中具有抑制腫瘤的特性。研究表明， 由EBV誘導的SSTR1所具有的表觀遺傳學抑制作用參與EB病毒相關性胃癌的發病機制。
Background and Aims: Epstein-Barr virus (EBV)-associated gastric cancer (GC) accounts for about 10% of all GCs. Accumulating evidences revealed aberrant rmethylation of tumor suppressor genes in EBV-associated GCs. However, the effect of EBV infection on the genome-wide aberrant DNA methylation remains unclear. We aim to profile the genome-wide EBV-associated hypermethylation in EBV-infected cells, to identify EBV-associated methylated genes and to elucidate their function in gastric carcinogenesis.
Methods: The cell model of gastric cancer AGS cells with or without stable EBV infection was used in this study. Genome-wide DNA methylation profiles were compared between AGS-EBV and AGS cells by high resolution Methyl-DNA immunoprecipitation microarry (MeDIP-chip) assay. EBV-associated methylated genes were classified according to gene ontology (GO). The novel EBV-associated methylated candidates were validated using bisulfite genomic sequencing (BGS), RT-PCR, and demethylation treatment. Biological function of one of the candidate genes (Somatostatin Receptor 1, SSTR1) was studied in vitro using gene knockdown and over-expression approaches simultaneously. Effects of SSTR1 expression on gastric cancer cell was measured by cell viability assay, colony formation assay, flow cytometry, wound-healing assay and invasion assay. Gene modulation by SSTR1 in human cancer pathways was assessed by cancer pathway PCR array.
Results: EBV infection was confirmed by EBER in situ hybridization. LMP2A expression was detected in AGS-EBV cells but not in EBV negative AGS cells. Expression and activity of DNMT3b was found to be significantly increased in AGS-EBV cells compared to AGS cells. Ectopic expression of LMP2A in AGS enhanced the expression and activity of DNMT3b. MeDIP-chip profiling identified a total of 1,065 genes differentially methylated by EBV infection (fold changes ≥2, P < 0.05) (fold-changes 2.4365.2). Gene ontology analysis indicated the enrichment of hypermethylated genes involving in important KEGG pathways. Notably, in addition to higher methylation levels confirmed by BGS, six novel hypermethylated genes (MDGA2, IL15RA, SCARF2, EPHB6, SSTR1 and REC8) were down-regulated in AGS-EBV cells as compared with AGS cells. Furthermore, demethylation treatment increased transcription levels of the six genes in AGS-EBV cells.
The biological function of SSTR1 gene was further investigated. Knockdown of SSTR1 in GC cells increased cell proliferation (P < 0.05) and colony formation ability (P < 0.01), and markedly increased cells in S phase through regulating G1/S phase mediators. Overexpression of SSTR1 in PANC1 cell line further confirmed that SSTR1 indeed was a tumor suppressor gene. Analysis of SSTR1 regulation of cancer pathway demonstrated that SSTR1 exerted antiproliferative effect by inducing cyclin-dependent inhibitors (p15, p16, p21 and p27) and inhibiting cell divison cycle 25 homolog A (CDC25A) and Myc, resulting in cell cycle arrest in G1 phase and reduction of cell proliferation. SSTR1 also took part in proliferation by decreasing expression of apoptosis regulators. Moreover, SSTR1 significantly downregulated the expression of migration-related genes, including ITGA1, ITGA2, ITGA3, ITGB5, IL8, MMP1 and PLAUR. These findings suggest that SSTR1 inhibits proliferation and reduces cell migration/invasion in gastric cancer by deregulating genes invloved in the regulation of cell cycle, survival/apoptosis and migration.
Conclusions: EBV infection in AGS cells induces genome-wide aberrant hypermethylation of 886 genes which involved in important cancer-related pathways. EBV-associated methylation is mediated by activation of DNMT3b through LMP2A. We identified and functionally characterized a novel EBV-associated methylated gene SSTR1 which exerted anti-tumor properties in GC. Epigenetic silencing of SSTR1 associated with EBV infection contributes to the pathogenesis of EBV-associated GC.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Zhao, Junhong.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 129-141).
Abstract also in Chinese.
ABSTRACT --- p.i
摘 要 --- p.iv
Acknowledgements --- p.vi
Publications --- p.vii
Research articles --- p.vii
Conference abstracts --- p.viii
Table of contents --- p.x
list of tables --- p.xiii
list of figures --- p.xiv
list of abbreviations --- p.xvi
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- General introduction --- p.1
Chapter 1.2 --- Gastric Cancer --- p.3
Chapter 1.2.1 --- Eipdemiology of Gastric Cancer --- p.4
Chapter 1.2.2 --- Pathology of Gastric Cancer --- p.8
Chapter 1.2.3 --- Risk Factors for Gastric Cancers --- p.11
Chapter 1.3 --- Epstein-Barr Virus-associated Gastric Cancer (EBVaGC) --- p.15
Chapter 1.3.1 --- Historical Discovery and Harm of EBV --- p.15
Chapter 1.3.2 --- Molecular Biology of EBV --- p.16
Chapter 1.3.3 --- Latent and Lytic Infection of EBV --- p.18
Chapter 1.3.4 --- EBV Products --- p.18
Chapter 1.3.5 --- EBV-associated gastric cancer (EBVaGC) --- p.28
Chapter 1.4 --- EBV-induced Epigenetic Alteration in Gastric Carcinogenesis --- p.36
Chapter 1.4.1 --- Cytosine Methylation and CpG Island --- p.36
Chapter 1.4.2 --- DNA Methylation in Gastric Cancer --- p.39
Chapter 1.5 --- How to identify EBV-induced promoter methylation in gastric cancer --- p.45
Chapter 1.5.1 --- Methylated DNA Immunoprecipitation (MeDIP) --- p.48
Chapter 1.5.2 --- Combined Bisulfite Restriction Analysis (COBRA) --- p.49
Chapter 1.5.3 --- Bisulfite Genomic Sequencing --- p.49
Chapter 1.5.4 --- Pyrosequencing --- p.49
Chapter Chapter 2 --- Materials and Methods --- p.51
Chapter 2.1 --- Materials --- p.51
Chapter 2.1.1 --- Cancer Cell Lines and Culture Condition --- p.51
Chapter 2.1.2 --- Primary GC Samples --- p.52
Chapter 2.2 --- EBV Encoded Nuclear RNA (EBER) in situ Hybridization (EBER-ISH) --- p.52
Chapter 2.3 --- Western Blot Analysis --- p.53
Chapter 2.4 --- Plasmid and Transfection --- p.56
Chapter 2.4.1 --- Plasmid Construction and Extraction --- p.56
Chapter 2.4.2 --- Plasmid Transfection --- p.59
Chapter 2.5 --- Gene Expression Analysis --- p.59
Chapter 2.5.1 --- Purification of Total RNA (RNeasy Kit, Qiagen) --- p.59
Chapter 2.5.2 --- cDNA Reverse Transcription --- p.60
Chapter 2.5.3 --- Semi-Quantitative PCR --- p.62
Chapter 2.5.4 --- Quantitative Real-Time PCR (qRT-PCR) --- p.64
Chapter 2.6 --- DNMT1 and 3b Activity Assay --- p.64
Chapter 2.7 --- DNA Methylation Analysis --- p.64
Chapter 2.7.1 --- Genomic DNA Extraction --- p.64
Chapter 2.7.2 --- Genome-wide Profiling of EBV-associated DNA Methylation by MeDIP-chip --- p.65
Chapter 2.7.3 --- Bioinformatics Analysis --- p.66
Chapter 2.7.4 --- CpG Island Prediction and Analysis of the Targets’ Promoter Region --- p.66
Chapter 2.7.5 --- DNA Sodium Bisulfite Modification --- p.67
Chapter 2.7.6 --- Target Gene Methylation in GC Cell Lines --- p.67
Chapter 2.7.7 --- Bisulfite Pyrosequencing Analysis in GC Tissue Samples --- p.70
Chapter 2.8 --- Biological Function Analysis of SSTR1 --- p.72
Chapter 2.8.1 --- Cell Proliferation Assay for Stable Transfection --- p.72
Chapter 2.8.2 --- Colony Formation Assay --- p.72
Chapter 2.8.3 --- Cell Cycle Analysis Assay --- p.72
Chapter 2.8.4 --- Cell Migration Analysis --- p.73
Chapter 2.8.5 --- Invasion Analysis --- p.73
Chapter 2.8.6 --- Human Cancer Pathway Finder RT2 Profiler PCR Array Analysis --- p.74
Chapter 2.9 --- Statistical Analysis --- p.76
Chapter Charpter 3 --- results --- p.77
Chapter 3.1 --- EBV Infection in AGS-EBV Cell Model --- p.77
Chapter 3.2 --- Activation of DNMT3b in AGS-EBV Cells --- p.80
Chapter 3.3 --- LMP2A Induced DNMT3b Activity in AGS Cells --- p.82
Chapter 3.4 --- Genome-wide Profiling of DNA Methylation Associated with EBV Infection Using MeDIP-chip --- p.84
Chapter 3.5 --- EBV-associated Cancer Pathways Defined by EBV-associated Promoter Methylated Genes --- p.86
Chapter 3.6 --- CpG Hypermethylation and Transcriptional Silencing of EBV-associated Methylated Genes in AGS-EBV Cells --- p.88
Chapter 3.7 --- Bioinformatics Analysis of SSTR1 Using University of California Santa Cruz Genome Bioinformatics (UCSC) Database and CpG Island Searcher --- p.93
Chapter 3.8 --- COBRA Analysis of SSTR1 Promoter Methylation in GC Cell Lines --- p.93
Chapter 3.9 --- Frequent SSTR1 Hypermethylation was Associated with EBV Positive Primary Gastric Cancer --- p.96
Chapter 3.10 --- SSTR1 was Down-regulated in GC Cell Lines through RNA Interference --- p.103
Chapter 3.11 --- SSTR1 Knockdown Induced Cell Proliferation in GC Cell Lines --- p.105
Chapter 3.12 --- SSTR1 Knock-down Promoted Cells to Enter into S Phase --- p.108
Chapter 3.13 --- SSTR1 Knock-down Increased the Migration Ability of GC --- p.110
Chapter 3.14 --- SSTR1 Knock-down Promoted Cell Invasion --- p.112
Chapter 3.15 --- Ectopic Expression of SSTR1 Inhibited Proliferation and Clonogenicity in PANC1 Cancer Cells --- p.114
Chapter 3.16 --- Identification of Genes Modulated by SSTR1 --- p.116
Chapter Chapter 4 --- Discussion --- p.119
Chapter Chapter 5 --- Limitation of the study --- p.127
ConclusionS --- p.128
Reference --- p.129

碩士
國立中央大學
材料科學與工程研究所
105
Studying circulating tumor cells (CTCs) helps us understand the biology of metastasis. Monitoring CTCs counts in patients’ blood can also help us know if the treatment works or not. Most cancer patients were died because of distant metastases instead of primary tumor. Thus, early detection of CTCs could provide early diagnosis of metastases and even personalized therapy. Herein, we introduced a new CTC capturing substrate composed of Si hierarchical nanostructures (Pyramid NW) method to capture nasopharyngeal carcinoma (NPC) cell in vitro. According to the results, the pyramid microstructure increased the surface area because of its geometric shape. The amount of antibodies on the substrate also increase, enhancing the cell-binding ability of the substrate. The nanowire (NW) structure improved the cell adhesion by letting the cytoskeleton of the cells extend into the gaps between the NW, making pyramid NW structure an ideal substrate for CTC capture. The Epstein-Barr virus (EBV) DNA titers in NPC patient’ blood is usually higher than normal people. In recent years, Surface enhanced Raman scattering (SERS) has become an efficient technique for detecting DNA because of its single molecule level sensitivity and molecular specificity. In this study, our pyramid NW substrate can easily be transformed into a SERS substrate by dipping it into AgNO3 solution. The DNA detection limit can reach up to 10-13M and the calculated enhancement factor can achieve up to 107.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2021
O cancro oral é um problema de saúde pública, tanto nos países desenvolvidos como nos que estão em desenvolvimento e constitui uma importante causa de doença e morte. O vírus Epstein-Barr (EBV) apresenta um tropismo por linfócitos B e também por células epiteliais. Embora a infeção geralmente seja benigna, parece estar associada a vários linfomas e carcinomas que surgem na cavidade oral e em outros locais anatómicos. Nos últimos anos, tem sido investigada a relação com a carcinogénese oral, devido ao seu potencial oncogénico. Este trabalho teve como principal objetivo a realização de uma pesquisa sobre o papel da infeção pelo EBV na carcinogénese oral. Visou-se ainda tentar contribuir com informação que auxilie o médico a compreender a importância desta infeção no cancro oral. Foi realizada uma pesquisa através da base de dados da PubMed®, de artigos publicados nos últimos 10 anos, utilizando os termos MeSH: “Epstein-Barr Virus Infections” e “Mouth Neoplasms”. Os artigos a incluir foram selecionados com base no título e relevância do seu resumo. Na totalidade foram incluídos 29 artigos. A presença de EBV é comum em tecidos da cavidade oral. A prevalência de EBV em amostras orais varia amplamente entre os estudos. A positividade para a presença do EBV em carcinomas orais apresenta uma variação de estudo para estudo, numa proporção entre 30% e 90%. A infeção por EBV e o risco de cancro oral foi consistentemente e positivamente relatado. Investigadores acreditam que, o EBV provavelmente confere fenótipos malignos observados em casos tumorais positivos para EBV. A cavidade oral serve como o principal local de residência e transmissão do EBV. No entanto, outros estudos com amostras maiores são cruciais para confirmar o papel do EBV na patogénese do cancro oral.
Oral cancer is a public health problem in both developed and developing countries and is a major cause of illness and death. EBV has a tropism for B lymphocytes and also for epithelial cells. Although the infection is usually benign, it appears to be associated with a number of lymphomas and carcinomas that arise in the oral cavity and other anatomical sites. In recent years, the relationship with oral carcinogenesis has been investigated, due to its oncogenic potential. The main objective of this work was to carry out a research on the role of EBV infection in oral carcinogenesis. It was also intended to try to contribute with information to help the physician to understand the importance of this infection in oral cancer. A search was carried out through the PubMed® database, of articles published in the last 10 years, using the MeSH terms: “Epstein-Barr Virus Infections” and “Mouth Neoplasms”. The articles to include were selected based on the title and relevance of their abstract. 29 articles were included. The presence of EBV is common in tissues of the oral cavity. The prevalence of EBV in oral samples varies widely between studies. The positivity for the presence of EBV in oral carcinomas varies from study to study, in a proportion between 30% and 90%. EBV infection and the risk of oral cancer were consistently and positively reported. Investigators believe that EBV likely confers malignant phenotypes seen in EBV-positive tumor cases. The oral cavity serves as the main place of residence and transmission of EBV. However, other studies with larger samples are crucial to confirm the role of EBV in the pathogenesis of oral cancer.

Lou, Pak Kin.
Thesis (M.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 136-170).
Abstracts in English and Chinese.
Abstract --- p.i
Acknowledgements --- p.v
Table of Contents --- p.vi
List of Figures --- p.x
List of Tables --- p.xiii
List of Publications --- p.xv
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1. --- Aims of Study --- p.1
Chapter 1.2. --- Literature Review --- p.2
Chapter 1.2.1. --- Nasopharyngeal Carcinoma --- p.2
Chapter 1.2.1.1. --- Overview --- p.2
Chapter 1.2.1.2. --- Histopathology --- p.2
Chapter 1.2.1.3. --- Epidemiology --- p.3
Chapter 1.2.1.4. --- Etiology --- p.5
Chapter 1.2.1.4.1. --- Epstein-Barr Virus (EBV) Latent Infection --- p.5
Chapter 1.2.1.4.2. --- Environmental Factors --- p.5
Chapter 1.2.1.4.3. --- Genetic Factors --- p.6
Chapter 1.2.1.5. --- Molecular Pathogenesis --- p.7
Chapter 1.2.1.5.1. --- Chromosomal Alterations --- p.7
Chapter 1.2.1.5.2. --- NPC-associated Tumour Suppressor Genes --- p.7
Chapter 1.2.1.5.3. --- NPC-associated Oncogenes --- p.8
Chapter 1.2.2. --- Epstein-Barr Virus --- p.9
Chapter 1.2.2.1. --- Overview --- p.9
Chapter 1.2.2.2. --- Lytic and Latent Infection of EBV --- p.9
Chapter 1.2.2.3. --- EBV Latency Programs and Associated --- p.10
Malignancies --- p.11
Chapter 1.2.2.4. --- The Role of EBV in NPC --- p.12
Chapter 1.2.3. --- NF-kB Signalling Pathways --- p.12
Chapter 1.2.3.1. --- Overview --- p.12
Chapter 1.2.3.2. --- Pathway Components --- p.12
Chapter 1.2.3.2.1. --- NF-kB Subunits --- p.16
Chapter 1.2.3.2.2. --- Inhibitors of kB (IkBs) --- p.16
Chapter 1.2.3.2.3. --- IkB Kinases (IKKs) --- p.17
Chapter 1.2.3.3. --- NF-kB Activation and Signalling --- p.17
Chapter 1.2.3.3.1. --- The Canonical Pathway --- p.18
Chapter 1.2.3.3.2. --- The Non-canonical Pathway --- p.18
Chapter 1.2.3.3.3. --- Physiological Functions of NF-kB --- p.19
Chapter 1.2.3.4. --- NF-kB Signalling and Tumourigenesis --- p.20
Chapter 1.2.3.4.1. --- Oncogenic Activation of NF-kB in Hematological Malignancies --- p.20
Chapter 1.2.3.4.2. --- Oncogenic Activation of NF-kB in Solid and Epithelial Tumours --- p.22
Chapter Chapter 2 --- Material and Methods --- p.22
Chapter 2.1. --- Tumour Specimens --- p.24
Chapter 2.2. --- NPC Tumour Lines and Immortalized NP Cell Lines --- p.24
Chapter 2.2.1. --- Cell Lines --- p.24
Chapter 2.2.2. --- Xenografts --- p.27
Chapter 2.3. --- DNA Sequence Analysis --- p.27
Chapter 2.3.1. --- Genomic DNA Extraction --- p.27
Chapter 2.3.2. --- Polymerase Chain Reaction (PCR) --- p.28
Chapter 2.3.3. --- DNA Sequencing --- p.32
Chapter 2.4. --- RNA Expression Analysis --- p.32
Chapter 2.4.1. --- Total RNA Extraction and Reverse Transcription --- p.33
Chapter 2.4.2. --- Quantitative Real-time Polymerase Chain Reaction (QRT-PCR) --- p.35
Chapter 2.5. --- Protein Expression Analysis --- p.35
Chapter 2.5.1. --- Total Protein Extraction --- p.35
Chapter 2.5.2. --- Nuclear and Cytoplasmic Protein Isolation --- p.36
Chapter 2.5.3. --- Western Blotting --- p.39
Chapter 2.6. --- Immunohistochemical Staining --- p.41
Chapter 2.7. --- Statistical Analysis --- p.41
Chapter 2.8. --- Immunoprecipitation --- p.43
Chapter 2.9. --- Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay --- p.44
Chapter 2.10. --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.45
Chapter 2.11. --- Plasmid Preparation --- p.45
Chapter 2.11.1. --- Plasmids --- p.45
Chapter 2.11.2. --- Bacterial Transformation and Plasmid DNA Extraction --- p.46
Chapter 2.12. --- Transfections --- p.46
Chapter 2.12.1. --- Transient Transfection --- p.46
Chapter 2.12.2. --- Stable Transfection --- p.47
Chapter 2.13. --- Immunofluorescence --- p.47
Chapter 2.14. --- Cell Proliferation and Viability Analysis --- p.47
Chapter 2.15. --- Small Interfering RNA (siRNA) Knockdown --- p.49
Chapter 2.16. --- Expression Microarray --- p.49
Chapter 2.16.1. --- Agilent Oligonucleotide Microarray --- p.50
Chapter 2.16.2. --- Data Analysis --- p.51
Chapter Chapter 3 --- Activation of NF-kB Signals in NPC --- p.51
Chapter 3.1. --- Introduction --- p.52
Chapter 3.2. --- Results --- p.52
Chapter 3.2.1. --- Expression Pattern of NF-kB Subunits in NPC Tumour Lines --- p.55
Chapter 3.2.2. --- Distinct NF-kB Complexes in NPC Tumour Lines --- p.60
Chapter 3.2.3. --- Expression of NF-kB Subunits in NPC Primary Tumours --- p.67
Chapter 3.3. --- Discussion
Chapter Chapter 4 --- Alterations of NF-kB Components in NPC --- p.71
Chapter 4.1. --- Introduction --- p.72
Chapter 4.2. --- Results --- p.72
Chapter 4.2.1. --- Homozygous Deletion of IicBa and TRAF3 in NPC Tumour Lines --- p.76
Chapter 4.2.2. --- Mutation of TRAF2 and A20 in NPC Tumour Lines
Chapter 4.2.3. --- Aberrant Expression of Multiple NF-kB Signalling Components in NPC Tumour Lines --- p.80
Chapter 4.2.4. --- Expression of NF-kB Signalling Components in NPC --- p.85
Primary Tumour --- p.92
Chapter 4.3. --- Discussion --- p.99
Chapter Chapter 5 --- Identification of Downstream Targets for NPC-associated NF-kB Signalling --- p.99
Chapter 0.1. --- Introduction --- p.99
Chapter 0.2. --- Results --- p.100
Chapter 0.2.1. --- Target Genes Modulated by p50 --- p.100
Chapter 0.2.2. --- Functional Annotation of p50 Target Genes --- p.105
Chapter 0.2.3. --- Target Genes Modulated by RelB --- p.105
Chapter 0.2.4. --- Functional Annotation of RelB Target Genes --- p.105
Chapter 0.2.5. --- Functional Annotation of Genes Modulated by both p50 and RelB --- p.111
Chapter 0.3. --- Discussion --- p.118
Chapter Chapter 6 --- Functional Role of TRAF3 Inactivation in NPC --- p.118
Chapter 0.1. --- Introduction --- p.118
Chapter 0.2. --- Results --- p.118
Chapter 0.2.1. --- Effect of TRAF3 Restoration on NF-kB Activity --- p.119
Chapter 0.2.2. --- Effect of TRAF3 Expression on Cell Proliferation --- p.123
Chapter 0.2.3. --- TRAF3 Expression Modulates Interferon Transcription in NPC Cells --- p.128
Chapter 0.3. --- Discussion
Chapter Chapter 7 --- General Discussion --- p.132
Chapter Chapter 8 --- Conclusion
Chapter Chapter 9 --- References
Appendix --- p.136