MicroRNA analysis of the invasive margin of glioblastoma reveals drugable therapeutic targets in lipid metabolism pathways

Glioblastoma (GBM) is the most common type of adult brain tumour, representing 45.2% of all malignant brain and central nervous system tumours. It is also the most lethal, with a median post-therapy survival of 14 months, due to the high likelihood of tumour recurrence. Intratumour heterogeneity is thought to be a key contributor to GBM tumour recurrence, because treatments such as radiotherapy and chemotherapy typically only successfully target the core of the tumour. Due to its high visibility, this core is excised during surgery, but there is another more subtle region of the tumour, the invasive margin, which generally remains behind. This invasive margin is a mixture between normal brain and highly invasive tumour cells. If left over from surgery, these remaining tumour cells infiltrate the normal brain, and cause recurrence within 2cm of the original site. However, if surgeons are too vigorous in removing the invasive margin, they may cause unnecessary removal of healthy brain tissue, thereby causing other complications. This make developing specific methods to target the cancer cells in the invasive margin a priority area in GBM research. In order to specifically target the invasive margin, we need to have a way of differentiating it reliably from the other regions. In this thesis, I investigated the role that micro RNAs (miRNAs), a type of small non-coding RNA, play in this intratumour heterogeneity. miRNAs act as post-transcriptional regulators of gene expression and can simultaneously suppress multiple genes within the same pathway, which makes them potentially valuable routes for therapy development. I performed a miRNA microarray on tissue samples from three tumour regions (tumour core, rim, and invasive margin) and validated the results with qRT-PCR. I then performed computational predictions of gene targets of the differentially expressed miRNAs and validated them in cell lines and patient samples at both the mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) levels. I then performed a bi-phasic extraction of polar and non-polar metabolites and employed liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy techniques to profile the intracellular metabolome and lipidome of U87 cells overexpressing miR-619-5p and miR-4793-3p. Transwell invasion assays were performed to investigate the effect of each miRNA and its target on GBM cell invasion. In this work, I found that two miRNAs, miR-619-5p and miR-4793-3p, are significantly downregulated in the invasive margin. Moreover, these miRNAs control the expression of genes that are involved in regulating lipid metabolism pathways. A second key finding was that the knockdown or pharmacological inhibition of the miR-619-5p target, CPT2, also significantly reduced the invasion of the cells in the invasive margin. Because CPT2 lies in the lipid metabolism pathway, my findings indicate that lipid metabolism may present a possible vulnerability of the GBM invasive cell population, and the understanding the function of miRNAs in regulating lipid homeostasis in GBM may provide novel avenues for GBM therapy

MicroRNA regulation of glycolytic metabolism in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most aggressive and common malignant brain and central nervous system tumour. A well-known hallmark of GMB, and many other tumours, is aerobic glycolysis. microRNAs (miRNAs) are a class of short non-protein coding sequences that exert post-transcriptional controls on gene expression and represent critical regulators of aerobic glycolysis in GBM. In GBM, miRNAs regulate the expression of glycolytic genes directly and via the regulation of metabolism-associated oncogenic pathways, such as the PI3K/Akt signalling pathway. The aim of this review is to establish links between miRNA expression levels, disease grade and prognosis, and the glycolytic phenotype of GBM. In this review, the involvement of 25 miRNAs in the regulation of glycolytic metabolism of GBM is discussed. Seven of these miRNAs have been shown to regulate glycolytic metabolism in other tumour types. Further eight miRNAs, which have been shown to be differentially expressed in GBM, were also reported to play a regulatory role in glycolysis in other cancer types. Such miRNAs could serve as potential glycolytic regulators in GBM but require functional validation. This review concludes with presenting a number of glycolytic regulatory miRNAs that have demonstrated their therapeutic potential either alone or as adjuvants in GBM, despite the major challenges that have to be solved before miRNA-based therapies can widely be used for the treatment of GBM patients

Intratumour Heterogeneity in MicroRNAs Expression Regulates Glioblastoma Metabolism

While specific microRNA (miRNA) signatures have been identified in Glioblastoma (GBM), the intratumour heterogeneity in miRNA expression has not yet been characterised. In this paper, we reveal significant alterations in miRNA expression across three GBM tumour regions: the core, rim, and invasive margin. Our miRNA profiling analysis show that miR-330-5p and miR-215-5p were upregulated in the invasive margin relative to the core and rim regions, while miR-619-5p, miR-4440 and miR-4793-3p were downregulated. Functional analysis of newly identified miRNAs suggests their involvement in regulating lipid metabolism pathways. Subsequent liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy (LC-MS/MS) profiling of the intracellular metabolome and lipidome of GBM cells with dysregulated miRNA expression confirm the alteration in the abundance of metabolites associated with lipid metabolism pathways. The identification of regional miRNA expression signatures may underlie a metabolic heterogeneity within the GBM tumour, and understanding this relationship may open new avenues for GBM treatment

MicroRNA analysis of the invasive margin of glioblastoma reveals drugable therapeutic targets in lipid metabolism pathways

Glioblastoma (GBM) is the most common type of adult brain tumour, representing 45.2% of all malignant brain and central nervous system tumours. It is also the most lethal, with a median post-therapy survival of 14 months, due to the high likelihood of tumour recurrence. Intratumour heterogeneity is thought to be a key contributor to GBM tumour recurrence, because treatments such as radiotherapy and chemotherapy typically only successfully target the core of the tumour. Due to its high visibility, this core is excised during surgery, but there is another more subtle region of the tumour, the invasive margin, which generally remains behind. This invasive margin is a mixture between normal brain and highly invasive tumour cells. If left over from surgery, these remaining tumour cells infiltrate the normal brain, and cause recurrence within 2cm of the original site. However, if surgeons are too vigorous in removing the invasive margin, they may cause unnecessary removal of healthy brain tissue, thereby causing other complications. This make developing specific methods to target the cancer cells in the invasive margin a priority area in GBM research. In order to specifically target the invasive margin, we need to have a way of differentiating it reliably from the other regions. In this thesis, I investigated the role that micro RNAs (miRNAs), a type of small non-coding RNA, play in this intratumour heterogeneity. miRNAs act as post-transcriptional regulators of gene expression and can simultaneously suppress multiple genes within the same pathway, which makes them potentially valuable routes for therapy development. I performed a miRNA microarray on tissue samples from three tumour regions (tumour core, rim, and invasive margin) and validated the results with qRT-PCR. I then performed computational predictions of gene targets of the differentially expressed miRNAs and validated them in cell lines and patient samples at both the mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) levels. I then performed a bi-phasic extraction of polar and non-polar metabolites and employed liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy techniques to profile the intracellular metabolome and lipidome of U87 cells overexpressing miR-619-5p and miR-4793-3p. Transwell invasion assays were performed to investigate the effect of each miRNA and its target on GBM cell invasion. In this work, I found that two miRNAs, miR-619-5p and miR-4793-3p, are significantly downregulated in the invasive margin. Moreover, these miRNAs control the expression of genes that are involved in regulating lipid metabolism pathways. A second key finding was that the knockdown or pharmacological inhibition of the miR-619-5p target, CPT2, also significantly reduced the invasion of the cells in the invasive margin. Because CPT2 lies in the lipid metabolism pathway, my findings indicate that lipid metabolism may present a possible vulnerability of the GBM invasive cell population, and the understanding the function of miRNAs in regulating lipid homeostasis in GBM may provide novel avenues for GBM therapy