GFAP revisited : From biomarker to an isoform-specific function in glioma malignancy

Gliomas are a heterogenous group of primary brain tumours that are incurable to date. Median survival after diagnosis for patients with the most common and most malignant glioma, i.e. astrocytoma grade IV or glioblastoma multiforme, is only 14.6 months. Heterogeneity of glioma cells within a single tumour and their highly invasive character complicates the design of effective and targeted therapies. In this thesis we focused on the role of the cytoskeletal intermediate filament (IF) glial fibrillary acidic protein (GFAP) in glioma malignancy. GFAP is the most well-known IF glioma biomarker and is used to identify well-differentiated, lower malignant astrocytoma. However, in the developing and adult human brain GFAP is expressed in a subpopulation of neurogenic stem cells as well that is characterized by specific expression of the GFAPδ isoform that results from alternative RNA splicing. GFAPδ expression in glioma is associated with higher malignant astrocytoma which has led us to question the use of GFAP levels, without distinguishing between isoforms, as a biomarker of lower malignant and more differentiated astrocytoma.Indeed, in this thesis we describe that the use of GFAP as a marker of low malignant, more differentiated, and non-invasive cells can be deceiving as GFAP is expressed in a heterogenous population of glioma cells that contains high malignant, stem-cell like, and more invasive cells as well. With the analysis of GFAP isoform expression in astrocytoma and other types of glioma patient material we show that the relative expression of the alternative splice variant GFAPδ to the canonical variant GFAPα (the GFAPδ/α ratio) is increased in higher grade astrocytoma and that the GFAPδ/α ratio might be useful to distinguish prognostic subgroups of glioma patients. The investigation of GFAP isoform-specific functions in regulating glioma malignancy by mimicking the observed increase in the GFAPδ/α ratio in high grade astrocytoma in astrocytoma cell lines and the analysis of genome wide gene expression alterations shows that a high GFAPδ/α ratio directs astrocytoma gene expression to a more malignant profile. Using CRISPR/Cas9 technology to modulate GFAP alternative splicing we further investigated this profile. We show that a high GFAPδ/α ratio induces changes in cell-extracellular matrix (ECM) interactions that better equip glioma cells to invade the brain. The results in this thesis suggest that high GFAPδ/α ratio glioma cells are high malignant, less differentiated, high invasive stem-cell like glioma cells. In addition, we describe the expression of a new GFAP isoform, GFAPλ, in glioma that further emphasizes the importance of continuing research on GFAP isoform diversity in expression and function. In this thesis we describe the transition of GFAP as a biomarker of low malignant astrocytoma to an isoform-specific regulator of glioma malignancy, with potential to contribute to the identification of glioma prognostic subgroups and being therapeutically targeted for glioma treatment. Future studies are needed to further elucidate the role of GFAP isoform-specific IF network functions in glioma cell malignant behaviour and to evaluate the feasibility of targeting GFAP alternative splicing and the IF network for the treatment of glioma