Significance of filamin A in mTORC2 function in glioblastoma

Background: Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes. Methods: To further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed. Results: In addition to the inhibition of mTORC2 activity, we demonstrated significant alteration of actin distribution as revealed by the use of phalloidin staining. Furthermore, vinculin staining was altered which suggests changes in focal adhesion. Inhibition of cell migration and invasion was observed with PP242. Two major proteins that are associated with this mTORC2 multiprotein complex were found. Mass spectrometry identified one of them as Filamin A (FLNA). Association of FLNA with RICTOR but not mTOR was demonstrated. Moreover, in vitro, purified mTORC2 can phosphorylate FLNA likewise its known substrate, AKT. In GBM cells, colocalization of FLNA with RICTOR was observed, and the overall amounts of FLNA protein as well as phosphorylated FLNA are high. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decreased. This treatment also disrupted colocalization of Actin filaments and FLNA. Conclusions: Our results support FLNA as a new downstream effector of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA signaling pathway plays important roles in motility and invasion of glioblastoma cells.