Characterising protein arginine methylation in glioblastoma cell lines : implications for brain cancer

Abstract

Protein arginine methylation is a post-translational modification that plays important roles in cellular signalling, transcription and RNA splicing and transport. The enzymes responsible for arginine methylation, protein arginine methyltransferases (PRMTs), have been reported to be aberrantly expressed in a variety of cancers, including breast, lung, bladder, prostate, ovarian and mantle cell lymphoma. It has become evident that arginine methylation is also a regulator of the cell cycle and aberrant expression of PRMTs contributes to deregulation of the cell cycle, contributing to carcinogenesis. Grade IV Glioblastoma multiforme is one of the most aggressive types of brain cancer. The 5- year survival rate for patients with brain cancer is only 20%. One of the difficulties in treating brain cancers is the low penetration of drugs across the blood-brain barrier. Hence, it is important to develop novel chemotherapy drugs to treat brain cancer which may be used alone or in combination with existing treatments. Protein arginine methylation is a potential target for novel drug development. In this thesis, protein arginine methylation in glioblastoma cell lines was characterised and the effect of the inhibition of two PRMTs was determined. The results showed, that although there were differences in the asymmetric and symmetric dimethylation of individual proteins, there were no consistent differences in the overall level of methylation between the control (non-cancerous) glial and glioblastoma (cancerous) cell lines. However, there were increases in the protein expression of the individual PRMTs in the glioblastoma cell lines when compared to the control glial cell line. Protein expression of PRMTs 1, 2, 5, 6 and 8 were all upregulated in A172 cells, PRMTs 5, 6 and 8 were upregulated in U87MG cells and PRMTs 5 and 6 were upregulated in T98G cells. A172 and T98G cells also had less protein expression of PRMT3 and PRMT1, respectively. Mass spectrometry analysis identified proteins with methylated arginine residues. Several proteins were identified to contain differentially methylated arginine residues in the four cell lines. Among these proteins, methylated arginine residues identified in the proteins, tubulin and Mortalin, have a functional significance which has been implicated in neurological malformations or disorders and cancer development, respectively, confirming that arginine methylation is an important regulator of cellular function. A recently described inhibitor of PRMTs 1 and 8 (trans-2, 3- dimethoxy-I²- nitrostyrene) was used to determine the effect of PRMT1 and 8 inhibition in glial and glioblastoma cells. It was discovered that the PRMT1/8 inhibitor had a cytotoxic effect on control glial cells but a cytostatic effect on glioblastoma cells. The PRMT1/8 inhibitor caused a significant arrest in the S phase of the cell cycle in all cell lines except in the T98G cells. Cells treated with the PRMT1/8 inhibitor were more sensitive to UV exposure and took longer to recover according to cell number. This suggests that the PRMT1/8 inhibitor may be a potential novel therapeutic agent that could be used in combination with DNA damaging chemotherapy agents. In summary, protein arginine methylation and the expression of PRMTs differs between normal glial cells and glioblastoma cells. Control glial and glioblastoma cells were sensitive to the inhibition of PRMT1/8 in a PRMT1-dependent manner and this contributed to a delayed DNA damage response when the cells were exposed to UV. The newly gained insight supports that protein arginine methylation is a key regulator of carcinogenesis in glioblastoma and has identified PRMTs as a therapeutic target. The results may contribute to the future discovery of novel drugs that can be used for cancer treatment.

Date of Award	2019
Original language	English