Thesis (M.S.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains vii, 78 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 70-78).

Glioblastoma multiforme (GBMs) are the most common and severe form of malignant brain tumors. Despite recent advancement in the fields of surgical resection, radiotherapy and chemotherapy, the prognosis for patients with GBMs remains poor with the median survival rate of approximately a year. Recently, our laboratory has demonstrated the oncogenic role of cell cycle-related kinase (CCRK), a 42-kD protein kinase responsible for regulating cell growth in GBM carcinogenesis, suggesting that CCRK is a candidate oncogene in GBMs. Nevertheless, the regulation of CCRK expression and the cellular mechanism for its overexpression in GBMs remain elusive. Understanding the regulation of human CCRK expression in GBMs should therefore shed light on the development of better prognostic and therapeutic methods for this deadly disease. This study aims to characterize the human CCRK gene and the regulation of its expression in GBMs. We first characterized the 5’ upstream sequence of CCRK by in silico analysis, which revealed the absence of TATA box but the presence of three potential transcription factor binding sites for Sp1, c-Myc and CREB, and identified the transcription start site by 5’-RACE at 240 bp upstream of the start codon. In vitro analysis of the CCRK promoters revealed the presence of nucleotide polymorphisms in three high-grade glioblastoma cell lines U-87 MG, U-138 MG and U-373 MG, and the control fibroblast cell line CCD19Lu. Furthermore, three CpG islands within the CCRK promoter were identified and the CCRK promoter was hypomethylated in these cell lines. Sp1, c-Myc and CREB binding sites as well as the nucleotide polymorphisms on the CCRK promoter were further investigated. The results from electrophoretic mobility shift assay showed that these transcription factors interacted with the corresponding cis-regulatory elements on the CCRK promoter, removal of the potential Sp1 and c-Myc binding sites lowered the CCRK promoter activity by 46 – 66 % in vitro. In addition, mutations introduced to the nucleotide polymorphisms reduced the CCRK promoter activity by 62 – 81 % in U-87 MG cells and enhanced the CCRK promoter activity by 1.35-fold in U-138 MG cells, suggesting their importance in regulating CCRK expression. c-Myc is a proto-oncogene with its overexpression associated with a large variety of tumors. As c-Myc binding site was identified in the CCRK promoter, therefore, the effect of c-Myc on CCRK expression was examined. c-Myc overexpression resulted in significant enhancements in the CCRK promoter activity by 30.74-fold in U-87 MG cells, 26.5-fold in U-373 MG cells and 6.09-fold in U-138 MG cells. On the contrary, c-Myc knockdown reduced the CCRK promoter activity by 49 % in U-87 MG cells, 35 % in U-373 MG cells and 17% in U-138 MG cells. In summary, this study represents the first molecular characterization of the human CCRK gene and findings of this study would prime others for future research on the molecular pathogenesis of CCRK-mediated GBMs and for developing CCRK as a potential therapeutic and diagnostic target for GBMs and possibly other cancers. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

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Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

Made available in DSpace on 2015-12-10T14:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-17. Added 1 bitstream(s) on 2015-12-10T14:29:14Z : No. of bitstreams: 1 000851655.pdf: 1706170 bytes, checksum: 990d3d441221c8d5284639c96fcf57c8 (MD5) / Os tumores cerebrais primários mais comuns são denominados gliomas. Eles são definidos patologicamente pela presença de características histológicas e imuno-histoquímicas que evidenciam diferenciação glial. De acordo com a suposta linhagem de origem, eles são classificados como astrocitomas, oligodendrogliomas ou ependimomas. Dentre eles, os astrocitomas são os mais comuns e agressivos. O tratamento atualmente utilizado inclui remoção cirúrgica seguida de quimioterapia com temozolamida (TMZ) e radioterapia, porém sua eficácia é muito baixa devido à alta resistência das células tumorais. Buscando encontrar genes associados com a elevada resistência dos astrocitomas, realizamos um estudo anterior de expressão gênica diferencial utilizando uma coleção de genes de reparo de DNA. Nesta análise foram identificados sete genes significantemente superexpressos em glioblastoma multiforme (GBM), o tipo mais agressivo de astrocitoma. Estes genes são: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L e XRCC2. Através de RT-PCR quantitativo, avaliamos os níveis de expressão destes genes em um painel expandido de 54 casos clínicos de astrocitomas de diferentes graus de malignidade e em 5 linhagens celulares de GBM. Todos os genes analisados mostraram-se mais expressos nos astrocitomas, com exceção de RAD54L em amostras de astrocitoma de grau II. Além disso, a superexpressão dos 7 genes avaliada isoladamente não exerce influência direta na sobrevida dos pacientes. Evidenciou-se ainda a superexpressão mais acentuada de EXO1 e NEIL3, que foram selecionados para realização de ensaios funcionais de silenciamento, e avaliação do ciclo celular e taxas de apoptose/morte efetiva das células. Estes ensaios foram realizados com as linhagens celulares T98G e U138MG, que apresentaram maiores níveis de expressão destes genes. Nos ensaios funcionais, observamos que o silenciamento... / Gliomas are the most common type of primary brain cancers. They are pathologically defined by the presence of histological and immunehistochemical characteristics that evidence glial differentiation. According to the hypothetical cell of origin they are classified in: astrocytomas, oligodendrogliomas and ependimomas. Among them, astrocytomas are the more common and aggressive type. The treatment currently used for GBM includes surgical resection of tumor followed by chemotherapy with temozolamide (TMZ) and radiotherapy, but this protocol is still insufficient due to the high resistance of cancer cells. Searching for repair genes associated with the high resistance of astrocytomas, we developed a previous study of differential gene expression using a collection of DNA repair genes. In this analysis, we identified seven genes significantly overexpressed in glioblastoma multiforme (GBM), namely: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L and XRCC2. Using quantitative RT-PCR, we evaluated the expression of these genes in an expanded panel of samples with 54 clinical cases of different grade astrocytomas and five GBM cell lines. All genes showed expression significantly higher in astrocytomas, except RAD54L in grade II astrocytomas. Moreover, the overexpression of this 7 genes evaluated individually doesn't exert direct influence upon patient's survival rate. Remarkably, EXO1 and NEIL3 showed the higher fold changes and were chosen for functional silencing assays. This experiments were performed with T98G and U138MG cell lines that showed the higher expression levels among the GBM cell lines analyzed. In the functional assays, we observed that the silencing of EXO1 or NEIL3 doesn't induce changes in the apoptosis and cell death rates and doesn't change the distribution of cells in cycle. Beyond this, the silencing of this two genes doesn't sentisizes cells to ionizing radiation.

Glioblastoma multiforme (GBM) is among the most aggressive and mortal cancers of the central nervous system. Maximal safe surgical resection, followed by radiotherapy accompanied with chemotherapy is the standard of care for GBM patients. Despite this intensive treatment with conventional approaches, the management of GBM remains poor. The infiltrative nature of cancer cells makes the complete tumour removal by surgery virtually impossible. In addition, the blood-brain barrier’s (BBB) lack of permeability limits the number of effective chemotherapy drugs for GBM. Temozolomide (TMZ) is the most widely used chemotherapeutic agent for GBM because of its ability to pass the BBB. However, high systemic doses required to achieve brain therapeutic level, resulting in numerous side effects. The recurrence of GBM is almost inevitable due to the aforementioned shortcomings of conventional methods of treatment. Therefore, a great deal of effort has been focused on the development of new treatment methods capable of providing a high concentration of chemotherapy drug at the tumour site. Microspheres made from biodegradable polymers hold great potential to keep the chemotherapeutic agent intact within the carrier and locally deliver the drug over an extended period. However, the encapsulation of amphiphilic drug molecules such as TMZ within poly (d, l-lactide-co-glycolide) (PLGA) microspheres with conventional emulsion methods, oil-in-water (o/w), water-in-oil-in-water (w/o/w), is a major challenge. The extremely low encapsulation efficiencies obtained for TMZ-loaded PLGA microspheres using the aforementioned techniques (<7%) hampers the ability to scale up this process. Additionally, the injected microspheres to the tumour site tend to dislocate due to the cerebral flow which reduces the effectiveness of this localized drug delivery strategy. This study has focused on the development of a 3D bioprinted hydrogel-based mesh containing TMZ-loaded PLGA microspheres with high encapsulation efficiency (GlioMesh). To accomplish this, oil-in-oil (o/o) emulsion solvent evaporation technique was used to prepare PLGA microspheres loaded with TMZ. The poor solubility of TMZ in the external oil phase, liquid paraffin, resulted in obtaining encapsulation efficiencies as high as 61%. We then used the 3D bioprinting technology to embed TMZ-loaded PLGA microspheres into an alginate mesh. This provides the advantage of immobilizing the microspheres at the tumour site. Additionally, the flexibility and porosity of 3D bioprinted mesh allow for easy implantation and nutrients transportation to the brain tissue. The incorporation of polymeric microspheres within alginate fibres led to achieving an extended release of TMZ over 50 days. The functionality of GlioMesh in inducing cell cytotoxicity was evaluated by performing in vitro cell viability tests on U87 human glioblastoma cells. Higher cytotoxic effects were observed in the case of treatment with GlioMesh compared to the free drug because of the sustained release properties of our mesh. These data suggest that GlioMesh holds great promise to be used as an implant in the treatment of GBM. / Graduate / 2019-04-19

Glioblastoma is the most common primary brain tumour in adults. Despite the aggressive standard of care currently used, median patient survival following treatment is only 14 months. Innovative treatment options are needed for these patients. Recently, oncolytic viruses have emerged as promising immunotherapies for the treatment of solid tumours. Preliminary work in our lab has demonstrated that Farmington virus, a novel brain-safe oncolytic rhabdovirus, can be engineered to encode a tumour-associated antigen (TAA) to prime and boost antigen-specific adaptive immune responses. Since other rhabdoviruses share this boosting capacity, a heterologous rhabdovirus prime/boost regimen can be designed to combine two powerful oncolytics and a robust anti-TAA adaptive immune response. We evaluated Farmington’s ability to vaccinate against a self- glioblastoma antigen and two foreign glioblastoma-associated antigens. Farmington was able to vaccinate against the foreign antigens, leading to efficacy in prophylactic and therapeutic glioblastoma models. Additionally, treatment with heterologous rhabdoviruses demonstrated efficacy in an aggressive murine mammary carcinoma model. Herein, we demonstrate promising preliminary results for a novel glioblastoma therapeutic approach. Le glioblastome est la tumeur primaire la plus fréquente chez l’adulte. La survie moyenne des patients n’excède pas 14 mois malgré une prise en charge thérapeutique agressive. Par conséquent, la mise au point de traitements innovants et efficaces est une nécessité pour ces patients. Des avancées récentes ont mise en évidence l’intérêt des virus oncolytiques dans le traitement des tumeurs solides. Des travaux préliminaires réalisés au sein de notre laboratoire ont, en effet, démontré que le virus Farmington pouvait être modifié afin d’exprimer un antigène associé aux tumeurs (AAT), pour initier et potentialiser une réponse immunitaire adaptative spécifique. D’autres rhabdovirus possèdent des capacités de potentialisation immunitaire similaires et peuvent être utilisés en association avec le virus Farmington modifié pour amorcer et amplifier la réponse immunitaire oncolytique de l’hôte. Le but de ce projet était d’évaluer le potentiel du virus Farmington comme vaccin contre des antigènes tumoraux d’origine endogène ou exogène associés au glioblastome. Nos résultats ont montré que le virus Farmington a la capacité d’induire une réponse immunitaire prophylactique et thérapeutique contre les antigènes tumoraux exogènes dans des modèles de glioblastome. De plus, l’utilisation de rhabdovirus hétérologues s’est aussi révélée efficace pour le traitement de carcinome mammaire agressif chez la souris. Cette étude préliminaire apporte des résultats prometteurs pour le développement de nouvelles approches thérapeutiques efficaces dans le traitement du glioblastome.

Made available in DSpace on 2014-06-12T23:03:12Z (GMT). No. of bitstreams: 2 arquivo8791_1.pdf: 2283735 bytes, checksum: 1046687aa31d4b605b161ab475c9b576 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / O Glioblastoma Multiforme (GBM) é o tumor glial com maior grau de malignidade. Acomete principalmente os hemisférios cerebrais apresentando sintomas e sinais focais ou gerais, relacionados ao tamanho, localização e taxa de crescimento tumoral. Objetivo: Analisar a relação do déficit motor com a topografia do GBM. Método: Foram estudados 43 casos de GBM, referidos quanto à idade, sexo, localização e a síndrome motora. Resultados: o tumor predominou em adultos (média de 55 anos), sexo masculino (55,82%), localização frontal (aproximadamente 40%). A hemiparesia prevaleceu como distúrbio motor, somente não ocorrendo em 2 casos de lesão frontal, 2 temporais, 1 parietal, 1 occipital e 1 fronto-temporal. Conclusão: Os achados clínico-topográficos favorecem os efeitos infiltrativos (lesões extensas) como responsáveis pela síndrome motora em detrimento aos efeitos compressivos (lesões localizadas)

Glioblastoma (GBM) is an aggressive brain cancer with devastating outcomes and few effective treatments. Although immunotherapy has shown promise in treating a variety of cancers, it is still unclear if and how it can be effectively used in GBM. Elucidating this will require a better understanding of the mechanistic role of immune cells and their interactions in the GBM tumor microenvironment. This thesis utilizes recent technological developments in cancer genomics and imaging to study the mechanisms underlying immunotherapy and the tumor microenvironment. First, we will provide background on our current understanding of GBM, its immune microenvironment, as well as modern sequencing and imaging methods. Second, we will present a longitudinal study of GBM patients before and after treatment with PD-1 immunotherapy. Only a small fraction of GBM patients respond to this type of therapy, so we perform genomic, transcriptomic, and spatial analyses to compare the molecular features of these rare responders versus non-responders. We show that clinical response to PD-1 immunotherapy in GBM is associated with specific molecular alterations and immune infiltration profiles that reflect the tumor’s clonal evolution during treatment. The most common infiltrating immune cells in GBM are macrophages, which are implicated in a wide variety of pro-tumor and anti-tumor roles. We then focus on this specific immune population by analyzing single-cell expression data from GBM tumors. We identify a novel macrophage subpopulation characterized by expression of the scavenger receptor MARCO, which drives tumor progression in GBM and is altered over the course of PD-1 immunotherapy. Next, we demonstrate that the methods we have developed for GBM are applicable to understanding the tumor microenvironments of other cancers as well. We analyze a cohort of melanoma cases to show that transcriptomic and imaging features can be combined to create a biomarker that stratifies patients into different risk groups. Finally, while most of the image analysis described so far has utilized histopathology, we include two appendices where we demonstrate new ways to process and analyze Magnetic Resonance Imaging (MRI) in GBM.