Finding the brake: dissecting the RhoA pathway regulating glioma cell migration

Abstract

Cell migration is one of the hallmarks of cancer. Cancer cells can adopt two main migratory strategies displaying either a mesenchymal or amoeboid phenotype. Targeting cell migration presents an opportunity in improving treatment of invasive and migratory tumours, however the cellular mechanisms that control the cell migration phenotypes in high-grade gliomas, such as Glioblastoma multiforme, are still not fully understood. In previous work we used 2D and 3D migration assays to screen a panel of inhibitors targeting the actin polymerisation pathway, a key regulator of cell migration in glioma. Amongst RhoA pathway inhibitors, which are key negative regulators of migration, we have identified one compound, CCG-1423, that uniquely failed to inhibit migration. CCG-1423 induces a reduced expression of the adhesion protein CCN1 by blocking nuclear import of MKL-1, a transcriptional co-activator. We suggest that CCG-1423 induces a mesenchymal transition, which permits continued migration. To confirm the effect of CCG-1423 on CCN1 levels we used ELISA to detect secreted CCN1 levels in culture supernatants in treated and non-treated U251 and U87 cells. CCG-1423 significantly reduced CCN1 levels in supernatant of both cell lines compared to untreated controls. Further biochemical analysis was performed to establish levels of MKL-1 in treated versus untreated cells and to elucidate its association with other proteins. Preliminary data indicate an increase in MKL-1 concentration in treated cells, consistent with a build-up of MKL-1 in the cytosol due to lack of nuclear import, as previously reported. Further detailed characterisation of the RhoA signalling pathway in glioma will provide an understanding of the role of MKL-1 in mesenchymal migration and the effect of CCG-1423 on mesenchymal switching. We will next characterise the interactions between CCG-1423 and MKL-1, and use pharmacological and/or genetic manipulation to refine combination treatments to target both mesenchymal and amoeboid cell migration.