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published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Orientador : Valeria Valente / Banca: Cleslei Fernando Zanelli / Banca: Ana Lúcia Fachin Saltoratto / Resumo: 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... / Abstract: 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. / Mestre

Glioblastoma multiforme is the most common and lethal primary brain tumour in adults. Its aggressive and invasive phenotype makes it resistant to current standards of care, with a patient median survival following treatment of only 14 months. Potent and safe therapeutics are necessary to improve patient prognosis. Globally, efforts are being made in immunotherapies to combat such deleterious tumours. Preliminary work in the Stojdl lab has developed a novel oncolytic virus platform for brain cancer therapy that is non-toxic and exhibits potent anti-tumour efficacy. This platform is based on the rhabdovirus Farmington, identified for its potent oncolytic properties and engineering malleability. Herein, we begin to show our capability to discover and vaccinate against immunogenic neoepitopes derived from a mouse cancer mutanome. Engineering Farmington virus to express neoepitopes, allows for robust tumour specific immune proliferation following a prime vaccination. Overcoming problems of targeting self-antigen and antigen loss variants, a multi-neoepitope vaccine, presented here, is one of many alternative approaches to help combat cancer resistance. Despite achieving robust anti-tumour immunity by vaccination, selectivity of the tumour microenvironment remains an enormous challenge. Cumulative efforts in immunotherapy research will help drive novel therapeutics, like Farmington, into clinic and, ultimately, improve patient’s prognosis and quality of life.

The study evaluated the survival of 364 glioblastoma multiforme (GBM) patients who received different modalities of treatment in two Canadian tertiary care centres. Retrospective and prospective databases were utilized to do a retrospective population based cohort study. The thesis question was among treated GBM patients in Edmonton and Halifax; does the survival rate differ with introduction of concomitant temozolomide and radiation therapy (RT) versus non concomitant treatment? Our results indicate that concomitant temozolomide with radiation therapy and surgery was associated with longer survival in comparison to radiation therapy with surgery. We also found that age; surgical resection and shorter time to radiation therapy are important factors for longer survival. / Clinical epidemiology

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.