ID #541 Differential cell surface proteomics of Hisotne-3 G34-mutant glioma, with functional valodation of membrane proteins: Flotillin-1, HMGB1 and GLUT1/3.

Abstract

Paediatric high-grade gliomas (pHGGs) account for 10% of CNS tumours, although this is a small percentage, they are responsible for 40% of the fatalities, therefore pHGGs have been characterised as “aggressive”. Histone H3.3 mutations, K27M and G34V/R have been identified as the two most common recurrent somatic mutations in paediatric HGG (pHGG). These mutations are present in 50% of pHGG compared to only 1% in adult gliomas.

Previously, we isolated and analysed cell membrane proteins of H3-mutant and H3-wild type glioma cell lines using protein mass spectrometric analyses. We identified differential topography cell membrane proteins in H3-G34R/V mutated pHGG cells, in comparison to H3- wild type tumour cells in culture. Among the differentially expressed cell membrane proteins were Flotillin-1, HMGB1 and GLUT1/3. Flotillin-1 dysregulation is recognised as a hallmark of tumorigenesis, while HMGB1 is known to function as an oncogene. GLUT1 and GLUT3 are members of the facilitated glucose transporter family, are also upregulated in cancer.

We subsequently performed functional analyses, including immunofluorescence microscopy and western blotting, to assess the expression and localisation of these cell-surface proteins in H3 G34 mutated and H3-wild type glioma cell lines, alongside non-cancerous control astrocytes, in order to validate these as targets for developing novel therapeutics. Flotillin-1 expression was found higher in glioma cell lines compared to astrocytic controls. HMGB1 was significantly upregulated in H3.3-G34 mutant and H3-WT relative to astrocytes. Increased expression of GLUT3 protein rather than GLUT1, was also observed in glioma cell lines.

To assess functional relevance, we used the inhibitor PP121 to target Flotillin1 and Glutor to inhibit GLUT1/3 activity. MTT assays demonstrated decreased cell viability and proliferation following these inhibitions. Results from these cell membrane protein functional assays will be presented.