BACKGROUND:The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.RESULTS:We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.CONCLUSIONS:Our functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Objetivos. Determinar la influencia de los marcadores: antígeno Ki67 y del gen p53 como factores pronóstico independiente en la sobrevida de los pacientes con Glioblastoma Multiforme. Materiales y métodos. Se revisó un total de 150 casos de pacientes con diagnóstico preliminar de Glioblastoma Multiforme (GBM) atendidos en el Instituto Nacional de Enfermedades Neoplásicas, entre los años 2008 y 2013 y se seleccionaron 60 casos que cumplían con los criterios de inclusión requeridos, información clínico patológica y seguimiento adecuado. Resultados. La media de edad es de 51 años (8-73 años), conformado por 34 hombres (56.7%) y 26 mujeres (43.3%). La mediana de sobrevida global (SG) es menor en el grupo de pacientes que sobreexpresaron el antígeno Ki67 (>= 20%), frente a los pacientes que tuvieron niveles de expresión moderada del antígeno (>= 10%). (26.5 vs 40 meses). Asimismo, se evidencia que la SG es mayor en los pacientes que expresan positivamente el gen p53 (>20%), frente a los pacientes que no llegaron a expresarlo. (40 vs 30 meses). Conclusiones. Tanto la expresión del antígeno ki67 como la expresión del gen p53 se pueden determinar como factores pronóstico de la sobrevida de pacientes que hayan sido diagnosticados con GBM con el fin de mejorar la calidad de vida de estos pacientes dándoles la posibilidad de recibir un tratamiento más específico acorde a los valores de estos marcadores inmunohistoquímicos.Objectives.To evaluate the influence of markers: Ki67 antigen and p53 gene as independent prognostic factors in the survival of patients with Glioblastom Multiform. Materials and methods. A total of 150 cases of patients with a preliminary diagnosis of Glioblastom Multiform (GBM) treated at the National Institute of Neoplastic Diseases between 2008 and 2013 were reviewed and 60 cases were selected that satisfy the inclusion criteria required, clinical pathological information and adequate follow-up information. Results. The mean age is 51 years (8-73 years), made up of 34 men (56.7%) and 26 women (43.3%). The median overall survival (OS) was lower in the group of patients who overexpressed the Ki67 antigen (> = 20%), compared to the patients who had moderate levels of antigen expression (> = 10%). (26.5 vs. 40 months). As well evidenced that the OS is higher in patients who positively express the p53 gene (> 20%), compared to patients who did not express it. (40 vs. 30 months. Conclusion. Both, the expression of the ki67 antigen and the expression of the p53 gene could be determined as prognostic factors for the survival of patients who have been diagnosed with GBM in order to improve the quality of life of these patients giving them the possibility of receiving a more specific treatment according to the values of these immunohistochemically markers.

High grade astrocytomas are very progressive brain tumors. Glioblastoma multiforme is the most frequent and the most malignant type with very infiltrative phenotype of the tumor cells. Fibroblast activation protein FAP is a predominantly membrane bound prolyl peptidase bearing exo- and endopeptidase hydrolytic activities. FAP is known to play a role in wound healing, cell migration and invasion and its expression is linked to the pathogenesis of several malignancies. mRNA expression of FAP is upregulated in 48% of glioblastomas according to The Cancer Genome Atlas microarray data. The involvement of FAP in the pathogenesis of astrocytic tumors is largely unknown. The aims of this work are to analyse the expression of FAP in primary cell cultures derived from high grade gliomas and to analyse the influence of FAP on the growth, migration and invasion of glioma cells. Our ELISA and western blot results showed heterogenous expression of FAP in the studied glioma primary cell cultures and cell lines. Both enzymatic activities characteristic of FAP were detected in the primary glioma cell culture P11 with high expression of FAP. In these cells, FAP was present not only in the typical plasma membrane localization, but also in the cytoplasm as demonstrated by immunofluorescence staining. The P11 cells...

The long-term goal of the Laboratory of Biology of Cytoskeleton is to explain in molecular terms the role of microtubules in cellular processes and to characterize proteins associated with microtubules and its organizing centers (MTOCs). The main attention of our research is focused on -tubulin and its role in microtubule nucleation. We have shown previously that besides its localization on centrosomes, -tubulin is found on celullar membranes (Macůrek et al., 2008), plant kinetochores (Dryková et al., 2003) and marginal bend of embryonal chicken erytrocytes (Linhartová et al., 2002). g-Tubulin also interacts with protein tyrosine kinases, that can regulate microtubule nucleation (Sulimenko et al., 2006; Macůrek et al. 2008). There are growing evidence that centrosomal proteins can affect genetic stability of the cell. g-Tubulin is involved in the regulation of the cell cycle (Vardy et al., 2002) and can participate in activation of checkpoint mechanism controlling the integrity of DNA. Recently, it has been proved its interaction with nuclear protein ATR (Zhang et al., 2007) and Rad51 (Lesca et al., 2005). That's why g-tubulin has been connected with the process of cancerogenesis. Cancer cells also display the changes in expression of b-tubulin isotypes (Katsetos et al., 2003). Moreover, increased level of...

O GBM é o tipo mais agressivo de glioma, apresentando células indiferenciadas (CTGs), responsáveis pela proliferação, invasão e recidiva tumoral. Avaliamos o papel da proteína PrPC e seu ligante HOP na proliferação e autorrenovação de CTGs. Cultivamos linhagens de GBM humano em neuroesferas e geramos populações knockdown para PrPC e HOP. Observamos co-localização de PrPC e CD133 na superfície e sua internalização conjunta estimulada por cobre, sugerindo recrutamento de CD133 mediado por PrPC. O silenciamento de PrPC reduz a expressão de marcadores de células-tronco e autorrenovação, diminui a expressão de proteínas de adesão e afeta a migração celular. O silenciamento de HOP reduz a proliferação, recuperada com o tratamento com HOP em células PrPC+. A capacidade tumorigênica e proliferativa de neuroesferas knockdown para PrPC e/ou HOP in vivo é reduzida. Finalmente, um peptídeo de HOP que bloqueia a interação com PrPC inibe a proliferação e autorrenovação em células PrPC+, indicando potencial do complexo PrPC-HOP como alvo para terapias contra o GBM. / GBM is the most aggressive type of glioma, presenting undifferentiated cells (GSCs), responsible for proliferation, invasion and tumor recurrence. We evaluated the role of the PrPC and its ligand HOP in the proliferation and self-renewal of GSCs. We cultured human GBM lineages in neurospheres and generated knockdown populations for PrPC and HOP. We observed co-localization of PrPC and CD133 on the surface and their co-stimulated copper internalization, suggesting PrPC-mediated recruitment of CD133. PrPC silencing reduces the expression of stem cell markers and self-renewal, decreases adhesion proteins expression, and affects cell migration. HOP silencing reduces proliferation, recovered with HOP treatment in PrPC+ cells. The tumorigenic and proliferative capacity of neurospheres PrPC and/or HOP knockdown in vivo is decreased. Finally, a HOP peptide which blocks PrPC-HOP interaction inhibits proliferation and self-renewal in PrPC+ cells, indicating PrPC-HOP complex potential as a target for therapies against GBM.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumour in adults, as well as the most common. The current standard therapy is maximal safe resection, followed by radiotherapy in combination with the alkylating agent, temozolomide. Despite this multi-model treatment approach, median survival is just 14.6 months and new therapies are urgently needed. Glioblastoma stem cells (GSCs) are a highly tumourigenic subpopulation of GBM cells believed to promote therapeutic resistance along with angiogenesis and metastasis. Cancer stem cells share crucial characteristics with normal stem cells such as their ability to self- renew, maintain proliferation and differentiate multi-potently. One strategy to target GSCs is to force them to differentiate into post-mitotic cells, as this would cause them to lose their long-term repopulation potential and would therefore limit tumour growth. In this study, I investigated if the transcription factor ASCL1 could drive neuronal differentiation in GSCs. ASCL1 is a key regulator of neurogenesis in the developing CNS and is sufficient to reprogramme fibroblasts, astrocytes and induced pluripotent cells into neurons. However, ASCL1 also promotes proliferation, and in GSCs, it has been shown to drive tumourigenesis by upregulating Wnt signaling. Given its role in two opposing functions, ASCL1 is tightly regulated by multi-site phosphorylation on serine-proline resides. It is phosphorylated when driving proliferation in cycling cells and is un(der)phosphorylated when activating the transcription of its downstream targets involved in differentiation. I found that endogenous ASCL1 was phosphorylated in GSC lines and hypothesized that dephosphorylating ASCL1 may drive differentiation. I tested this by overexpressing a phosphomutant form of ASCL1, and found that it drove cell cycle exit through the downregulation of Cyclin D2, CDK4 and CDK6. However, ASCL1 did not drive overt differentiation which suggests GSCs may not respond to differentiation cues.

by Yue Cheng. / "April 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 156-175). / 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.

Glioblastoma (GBM) is the most common intrinsic primary brain tumour. It is uniformly fatal, with median survival approximately 14 months. These tumours comprise a mixture of neural stem cell-like cells and more differentiated astrocytic cells. The former are thought to be responsible for tumour development and recurrence, and display self-renewal and differentiation capacity in vitro. Glioma stem cells (GSCs) are defined operationally by their capacity to initiate tumours on orthotopic transplant into immunocompromised mice. The Pollard lab has identified the neural developmental transcription factor Forkhead Box G1 (FOXG1) as the most consistently overexpressed gene in GBM-derived neural stem (GNS) cells compared to their genetically normal neural stem (NS) cell counterparts. Here we explore the function and critical downstream effectors of FOXG1 in NS and GNS cells. We find that, although FOXG1 is not essential for sustaining proliferation of NS or GNS cells (in vitro), high FOXG1 restricts astrocyte differentiation in response to BMP and can drive dedifferentiation of postmitotic astrocytes. We identify a potential cooperation with SOX2. ChIP-Seq and RNA-Seq were used to define transcriptional targets. FOXG1 directly controls critical cell cycle regulators FOXO3 and FOXO6 (two forkhead family proteins), as well as the proto-oncogene MYCN and key regulators of both DNA and chromatin methylation, including TET3 and CHD3. Pharmacological inhibitors of MYC block FOXG1-driven de-differentiation, whereas Vitamin C and 5-azacytidine – agents that disrupt DNA and chromatin methylation – can facilitate de-differentiation. CRISPR/Cas genome editing was used to genetically ablate the cell cycle inhibitor FOXO3, or remove the FOXG1-bound cis-regulatory region. These data suggest direct transcriptional repression of FOXO3 by FOXG1 may drive cells into cycle. We conclude that high levels of FOXG1 in GBM limit astrocyte differentiation commitment by direct transcriptional control of core cell cycle regulators and DNA/histone methylation.

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