An investigation into the roles of aspartate/glutamate transporters in glioma

Rana, Himani (2024). An investigation into the roles of aspartate/glutamate transporters in glioma. University of Birmingham. Ph.D.

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Abstract

Sadly, only 25% of patients diagnosed with glioblastoma (GBM) survive for longer than 12 months. GBM, like most solid tumours, is characteristically hypoxic, which leads to a metabolic rewiring in cancer cells. Interestingly, amongst other metabolic changes, aspartate
becomes a limiting metabolite in hypoxia. Aspartate is imported into the cell via aspartate/glutamate transporters located on the plasma membrane; these are specific members of the SLC1 transporter family. The cytosolic and mitochondrial aspartate pools are maintained via the activity of SLC25A12 and SLC25A13, which are mitochondrial aspartate/glutamate transporters. Thus, this thesis began by studying both the plasma membrane and mitochondrial aspartate/glutamate transporters as a panel.

Bioinformatic analysis of the expression of aspartate/glutamate transporters in different glioma grades identified upregulation of SLC25A13 and SLC1A3 in GBM. Based on this, we assessed for hypoxia-induced changes of aspartate/glutamate transporter expression in human GBM cell lines. This revealed a marked downregulation of the mitochondrial aspartate/glutamate transporters in hypoxia. Leading on from this observation, the hypoxic regulation of SLC25A12 and SLC25A13 was investigated. We found the downregulation is not a result of hypoxia-induced endoplasmic reticulum stress. Instead, our studies identified a potential downregulation of SLC25A12 in hypoxia via HIF-1, the major regulator of the metabolic adaptation to hypoxia.

As this is not the case for SLC25A13, the impact of acute loss of the transporters on HIF-1α, the O2 sensitive subunit of HIF-1, was explored. This revealed SLC25A13 loss stabilises HIF1α and SLC25A12 loss reduces this stabilisation. Based on observations throughout these studies, we hypothesised a post-translational modification of SLC25A13 in hypoxia and investigated this possibility via genetic modification of the cells, immunoblotting and bioinformatic analysis. Our data supported this hypothesis although further investigations are required.

Additionally, an investigation into the impact of acute and chronic loss of the mitochondrial aspartate/glutamate transporters on glioma cell proliferation identified acute loss results in a larger phenotype than chronic loss, and SLC25A13 loss had a greater impact than SLC25A12. Moreover, immunoblotting experiments revealed a potential increased reliance on the activity of the plasma membrane aspartate/glutamate transporters with acute SLC25A12 and SLC25A13 loss.

Lastly, the functional differences between SLC25A12 and SLC25A13 in normoxic and hypoxic glioma cells was investigated using stable isotope tracing. Unlike reports in the literature, our data discovered an alternative working cell model. Acute loss of SLC25A12 decreased lactate secretion which is indicative of a reduced cytosolic NADH pool. This reflects continued MAS activity in the absence of SLC25A12. Moreover, with acute SLC25A13 loss we identified a potential compensation of MAS activity with the SLC25A1 transporter. Interestingly, acute loss of SLC25A12, not SLC25A13, resulted in an increased reliance on an alternative carbon source for nucleotide production in hypoxia. This suggests SLC25A13 is the major transporter of the malate-aspartate shuttle, and SLC25A12 is functionally important for the maintenance of aspartate availability.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):