INTRODUCTION: A highly invasive phenotype is a hallmark of the malignant process in Glioblastoma multiforme, which remains a poorly understood field. The diffuse and infiltrative nature of these cancers presents a need for novel, anti-migratory treatment to prevent tumour cells migrating to healthy parts of the brain and to improve the success of the current standard care. The actin polymerization pathway is part of a complex set of cellular mechanisms, which allows cell migration in 2D and 3D. Microarray data has identified a family of genes involved in this pathway known as the ARHGAPs. The ARHGAPs encode a group of proteins called Rho GTPase activating proteins (Rho GAPs), which interact with Rho GTPases; a collection of molecular switches within the signal transduction pathways in eukaryotic cells. They are primarily known for their role in regulating the actin cytoskeleton and, therefore, are involved cell migration. Five members of the ARHGAP family were of particular interest as their expression appeared deregulated in response to treatment with the anti-migratory drug Bio-Indirubin (BIO). Two-D and 3D migration assays were carried out to validate the microarray data and investigate the role of these genes in cell migration further. METHOD: Tumour spheroids were generated from established adult glioma cell lines, U87 and U251, and embedded into collagen. The spheroids were then treated with a panel of anti-migratory drugs for 72 hours. These drugs included CCG-1423 and GSK429286A, two actin polymerization inhibitors, along with Bio-indirubin and LiCl. The spheroids were imaged at 24 hour intervals and the migration index (MI) for each spheroid was calculated. A protocol for immunohistochemistry established at Leeds University was applied and the spheroids were stained for expression of the ARHGAP proteins. In addition, immunofluorescence staining was also performed using the same ARHGAP antibodies to detect expression levels in the cell lines after random migration in 2D. RESULTS: In both U87 and U251, different expression levels of the ARHGAP candidate genes were observed. In the immunofluorescence assays, cytoplasmic protein localization of the proteins were detected, which either increased or decreased in response to drug treatment with Bio-Indirubin. For one of the ARHGAP proteins, a significant increase in expression level was observed, with evidence of its role as a negative regulator of cell migration. All of the results from the immunofluorescence study correlated with the previous microarray analysis that presented the ARHGAPs as potential targets. The results from the immunohistochemistry study will also be discussed. CONCLUSION: From our preliminary results we have established that the members of the ARHGAP family have very different roles in the actin polymerization pathway and cell migration. These contrasting roles have presented an approach in which we can target cell migration. There is scope to target cell migration by overexpression of the anti-migratory candidates and silencing of the pro-migratory candidates and for the development of novel small molecule inhibitors to improve treatment and survival in glioma patients.
|Number of pages||1|
|Publication status||Published - Jan 2017|
|Event||British Neuro-Oncology Society Conference: Trials, Technologies and T-Cells - Leeds, United Kingdom|
Duration: 29 Jun 2016 → 1 Jul 2016
https://www.bnos.org.uk/events/bnos-conference/ (Link to Conference Report)