GFAPδ/GFAPα ratio directs astrocytoma gene expression towards a more malignant profile

Abstract

Astrocytomas are the most common malignant brain tumours and are to date incurable. It is unclear how astrocytomas progress into higher malignant grades. The intermediate filament cytoskeleton is emerging as an important regulator of malignancy in several tumours. The majority of the astrocytomas express the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Several GFAP splice variants have been identified and the main variants expressed in human astrocytoma are the GFAPα and GFAPδ isoforms. Here we show a significant downregulation of GFAPα in grade IV astrocytoma compared to grade II and III, resulting in an increased GFAPδ/α ratio. Mimicking this increase in GFAPδ/α ratio in astrocytoma cell lines and comparing the subsequent transcriptomic changes with the changes in the patient tumours, we have identified a set of GFAPδ/α ratio-regulated high-malignant and low-malignant genes. These genes are involved in cell proliferation and protein phosphorylation, and their expression correlated with patient survival. We additionally show that changing the ratio of GFAPδ/α, by targeting GFAP expression, affected expression of high-malignant genes. Our data imply that regulating GFAP expression and splicing are novel therapeutic targets that need to be considered as a treatment for astrocytoma.

Importance of GFAP isoform‐specific analyses in astrocytoma

“…In short, studies that analyzed astrocytoma homogenates neither generate consistent results on the correlation of GFAP expression to astrocytoma malignancy grades and additional analyses in larger cohorts are needed. In our own previously published study in which we used RNA sequencing data of 310 patients available at TCGA we do show a strong decrease in general GFAP mRNA levels in grade IV compared to grade II and grade III astrocytoma (Stassen et al, ).…”

“…These cells are present at lower numbers in low‐grade astrocytoma and could potentially induce progression into higher malignancy grades. Differences in malignant behavior of cells with a high and low GFAPδ/α ratio support this hypothesis (Moeton et al, ; Stassen et al, ) and future studies should focus on unravelling the isoform‐specific function in astrocytoma malignancy. In conclusion, information is lost when the expression of different GFAP isoforms is neglected and can be deceiving when GFAP is used to determine the differentiation state of a cell in experimental and clinical settings.…”

“…We recently showed that quantification of the relative level of GFAPδ to GFAPα, the GFAPδ/GFAPα ratio, using RNA sequencing data obtained from the cancer genome atlas (TCGA) indeed indicates that low‐ and high‐grade astrocytoma express different combinations of GFAP variants. In astrocytoma grade IV, the GFAPδ/GFAPα ratio was significantly higher compared to grade II and III (WHO 2007; Stassen et al, ).…”

GFAP alternative splicing regulates glioma cell–ECM interaction in a DUSP4‐dependent manner

“…We then continued to analyze upstream regulators of the MAPK‐signaling pathway that have been linked to tumor cell malignant behavior and also regulate the cell's interaction with the ECM (51–59); they were previously shown to be regulated by changes in the GFAP network (14, 16), laminin‐111 and its integrin receptors. Laminin subunit α1 ( LAMA1 ), which encodes the laminin α1 chain, forms the ECM molecule laminin‐111 together with the β1 and γ1 chain of laminin ( Fig.…”

“…To knock down GFAP isoforms, lentiviral vector transductions containing short hairpin RNA (shRNA)–targeting GFAP‐α (GFAP‐α – ), shRNA‐induced knockdown of all GFAP isoforms (panGFAP; panGFAP), and nontargeting shRNA control (NTC) were performed to generate stable GFAP‐modulated glioma cell lines as previously described in refs. 14 and 15.…”