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

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

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Thesis (Ph. D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Biochemistry. Bibliography: leaves 147-171. Also available on the Internet.

Glioblastoma multiforme (GBM) is the most malignant form of brain cancer. Therapies targeting glioblastoma have not consistently been able to give those diagnosed the best prognosis. Treatments that directly infiltrate into the tumor are highly sought after. Indirubins have been used to treat various types of cancers and are a promising avenue for future glioma research. In the current study, we further researched several key GSK-3 inhibitors, BIO (an indirubin) and CHIR99021, in addition to LiCl, to see their effects on the translocation of β-catenin to the nucleus, and the invasion and migration of cells in both a sphere assay and an aortic ring assay. Here we studied anti-invasive therapies that may have a future role in GBM treatment. It is thought that combining conventional treatments with anti-invasive therapies will create cytotoxicity in and reduce migration of the tumor. Three types of cells were used throughout the experiments: HBMEC, HUVEC, and U251 glioma cells. We reported that GSK-3 inhibitors might have a valuable role in the treatment of GBM. The selected inhibitors (BIO, CHIR99021, and LiCl) all were shown to lessen cell migration and invasion in vitro in a range of assays and in all cell lines tested. All inhibitors tested cause a dose-dependent, reversible inhibition of glioma cell invasion in spheroid assays. BIO was shown to cause a rapid upregulation of total and nuclear β-catenin. BIO, at higher concentrations, also created a toxic environment for cells, sometimes killing them. This shows that a more in-depth experiment involving different BIO concentrations is needed to test the optimal concentration for treatment. Each of the experimented GSK-3 inhibitors also showed a change in the junctions between cells. NaCl as a control showed normal, spikey, junctions, while CHIR99021 and BIO caused the junctions to become more smooth. This suggests that GSK-3 inhibition has a role in either maintaining the ECM and/or in communication between cells. Also in this assay, there was a heterogeneity between cells treated with the same inhibitor and in the same dish, indicating that not all cells respond to each drug the same way. The reasons for this are not known and further investigation is required. A new construct was also made to report β-catenin transcriptional coactivation using luciferase expression as the reporter in response to these selected GSK-3 inhibitors.With the combined results of these experiments, we concluded that GSK-3 inhibitors may be a promising approach to the treatment of GBM. Further investigation is required before any treatments can be administered to those diagnosed.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system (CNS) that exhibits considerable variation in its clinical course. Recently, the World Health Organization (WHO) published a classification system for tumors of the CNS that combines histological features with molecular parameters to determine tumor grade. The incorporation of molecular biomarkers that carry both prognostic and predictive value adds another level of objectivity to the glioma grading system and will help guide clinical decision. As such, the assessment of biomarkers has become an integral part of tumor evaluation in neuro-oncology. This curriculum will discuss the clinical relevance of the most recently studied biomarkers with prognostic and predictive value in the evaluation of GBM. Biomarkers regularly used for the assessment of GBM include the IDH mutations, MGMT methylation status, and EGFRvIII. Furthermore, this review will offer a perspective on experimental approaches currently under investigation for treatment of GBM. LITERATURE REVIEW FINDINGS: MGMT methylation of the promoter region is associated with better treatment response from temozolomide (TMZ), an alkylating therapeutic. Treatment benefit was most prominent in the elderly population and therapy should be individualized for that age group. Patients with GBM characterized by IDH1/IDH2 mutations carry a better overall prognosis primarily due to their higher sensitivity to chemo- and radiotherapy. The prognostic value of EGFRvIII remains controversial, although it may be associated with a worse prognosis. Nonetheless, EGFRvIII provides an ideal target for targeted molecular therapies as it is only found on tumor cells. PROPOSED METHODS: A curriculum aimed at educating primary care providers (PCPs) about the most clinically significant biomarkers in GBM will be developed. The curriculum will be in a PowerPoint format, and the hour-long lecture will be presented at continuing medical education national conferences. A pre- and post-test consisting of the same 10 multiple- choice questions will be administered on a voluntary basis to help evaluate knowledge acquisition from the curriculum. Results will be evaluated with a paired t-test analysis. The tests will be will be administered through Poll Everywhere, a smartphone survey application. CONCLUSION: There is increasing evidence to suggest that therapies should be individualized according to specific biomarkers with predictive value. PCPs are in a position where they are often the first providers to suspect the diagnosis of a brain tumor. Therefore, it is imperative for PCPs to be aware of the latest development in the field of neuro-oncology so that they may appropriately counsel patients.

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Das Glioblastoma multiforme gehört zu den ZNS-Tumoren neuroepithelialen Ursprungs. Es zeichnet sich durch ein multiformes Zellbild, einen geringen Differenzierungsgrad und eine schnelle Krankheitsprogression aus. Trotz mikrochirurgischer Entfernung und anschließender Radiochemotherapie entwickeln die Patienten im Durchschnitt nach 7 Monaten einen Rezidivtumor und haben eine mittlere Überlebenszeit von 14,6 Monaten. Die Rezidivneigung stellt somit ein großes Problem in der Behandlung von Glioblastompatienten dar. In früheren Arbeiten konnte nachgewiesen werden, dass die Rezidivtumore eine andere Zellzusammensetzung und auch ein aggressiveres Wachstumsverhalten als deren Primärformen aufweisen. Ziel dieser Arbeit war es, zu prüfen ob mittels 2D-Gelelektrophorese und anschließender MALDI-TOF-Massenspektrometrie Unterschiede im Proteinexpressionsmuster zwischen Gewebeproben vom Primärtumor eines Glioblastoms WHO Grad IV und dem korrespondierendem Rezidivtumor eines Patienten detektierbar sind. Hierbei wurden 43 Proteine als differentiell exprimiert erkannt, von denen mit Hilfe der MALDI-TOF-Massenspektrometrie sechs genauer charakterisiert wurden. Vier der sechs Proteine waren im Rezidivtumor erhöht: EnoylCoA-Hydratase, ATP-Synthase Untereinheit d, Tropomyosin alpha-3-Kette Isoform 2 und Cathepsin D. Die anderen zwei waren im Rezidivtumor niedriger ausgeprägt: Nukleosid-Diphosphatkinase A und L-3-Phosphoserin-Phosphatase. Eine weitere Untersuchung mittels Western-Blot-Analyse bestätigte, dass Cathepsin D (als eines der sechs charakterisierten Proteine) tatsächlich auch in den Rezidivtumoren dreier weiterer Patienten stärker exprimiert war als in den korrespondierenden primären Glioblastomen.