Investigating HK2 as a potential therapeutic target in glioblastoma
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AbstractGlioblastoma is the most common high-grade primary brain tumour in adults. Current treatments have limited success with average survival comprehensively low. Effective drug treatments are hindered predominantly by the complex genetic background of glioblastoma heterogeneity, as such there is a compelling need for the development of effective therapeutics. An approach is to target abnormal metabolic pathways that are universally dysregulated in glioblastoma. The identification of potential targets and the development of therapeutic agents is crucial for the advancement of treatment. Hexokinase 2 (HK2) has a prominent role in glycolysis, acting as the rate-limiting step in the pathway. HK2 is highly expressed in many cancers and its role as the rate-limiting step of glycolysis may potentially contribute to tumour growth. Overexpression of HK2 has also been associated with drug resistant phenotypes, in parallel, its inhibition has improved the effectiveness of anticancer agents, suggesting HK2 as a potential therapeutic target. The role of methylation and its association with expression levels of HK2 was determined in glioblastoma fresh frozen biopsies and patient-derived cultures, through pyrosequencing and quantitative PCR. CRISPR knockout was utilised to investigate the effect of inhibiting HK2 on proliferation and to determine the role of HK2 in chemoresistance. The anti-proliferative effects of HK2 inhibitors 3-bromopyruvic-acid (3-BPA) and metformin were investigated via cytotoxic assays and FACS analysis was used to determine their mechanism of action. Additionally, downstream expression changes were investigated via expression profiler arrays, across 84 key genes involved in the regulation and enzymatic pathways of glucose metabolism. Hypomethylation was demonstrated in all biopsies (n=100) and cultures (n=15) compared to normal brain tissue; with average methylation of 4.6% compared to 26% respectively (p<0.0001) determined across 15-CpGs. A significant increase (p<0.0001) in HK2 expression was discovered, ranging between 6 to >1000- fold change in all biopsies and cultures compared to normal brain tissue and a strong correlation between hypomethylation and increased level of expression (p<0.0001) was established. Furthermore, elevated levels of HK2 revealed notably poorer survival outcomes. Sensitivity to 3-BPA in glioblastoma cultures was associated with elevated expression of HK2. Significantly different levels of apoptosis were observed with average levels 38% greater in high HK2 expressing cultures (p=0.0014). A significant growth rate reduction (p<0.007) was demonstrated in HK2-KO cultures compared to parent cultures. Importantly sensitivity to both metformin and TMZ was also significantly increased (p<0.0001) in response to HK2-KO, with substantial reduction in both ID50 values and cell survival. Notably, HK2-KO cultures yielded greater synergistic effects with metformin and TMZ combination treatment. Array data revealed significant downstream gene expression alterations (p<0.005) with HK2-KO and with the supplementation of both 3-BPA and metformin, where >50% genes demonstrated reduced levels of expression compared to the corresponding parent/non-treated cultures. This study demonstrates the predominant role of HK2 within the glycolytic pathway, with overexpression potentially key in driving the genetic alterations downstream. This study also verifies a strong correlation between increased expression of HK2 and hypomethylation, additionally highlighting the impact HK2 has on inferior patient prognosis. HK2-KO revealed considerable ubiquitous reductions in downstream gene expression compared to glioblastoma biopsy tissue and parent cultures. Additionally, an increase in drug sensitivity was depicted with the loss of HK2 signifying the potential of targeting HK2 as a novel therapy in a significant subset of glioblastoma.
PublisherUniversity of Wolverhampton
TypeThesis or dissertation
DescriptionA thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.
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