UCLA

ABSTRACT OF THE DISSERTATION

Characterizing the Mechanistic Target of Rapamycin Complex 2 Pathway in Glioblastoma Multiforme

by

Naphat Chantaravisoot

Doctor of Philosophy in Microbiology, Immunology, and Molecular Genetics

University of California, Los Angeles, 2015

Professor Fuyuhiko Tamanoi, Chair

The Mechanistic Target of Rapamycin Complex 2 (mTORC2) has been involved in multiple cellular processes that drive normal cells to maintain metabolic activities, survive, develop and proliferate properly. In diseased cells, mTORC2 plays an important role as a key regulator in tumorigenesis, promoting cell growth, supporting their irregular or metastatic abilities. In this dissertation, I focus my attention on glioblastoma multiforme (GBM) which 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.

My work has elucidated the functions of mTORC2 especially in the regulation of motility that glioblastoma cells use to support their highly migratory and invasive characters. Inhibition of mTORC2 by PP242, an ATP-competitive mTOR kinase inhibitor, blocks cell proliferation, disrupts focal adhesion, and alleviates cell migration and invasion. In addition, the treatment of RICTOR siRNA knocks down mTORC2 activity and significantly alters actin distribution as revealed by phalloidin staining. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal rearrangement and locomotion, I further affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Two major proteins that are associated with this mTORC2 multiprotein complex were identified as Filamin A (FLNA) and Myosin-9 (MYH9). Characterization of mTORC2 and its binding partners was performed to clarify their localization inside the cells under normal or inhibitory conditions. I performed the complex dissociation experiment to show that FLNA and MYH9 bind to RICTOR, not mTOR. Colocalization of FLNA with mTOR, RICTOR and MYH9 was observed suggesting that all of them are associated and physically located nearby.

In addition, my work demonstrated that the overall amounts of FLNA protein as well as phosphorylated FLNA are high compared with other cells. This is later found to be due to hyperactivated mTORC2 in GBM. FLNA can be phosphorylated in vitro by purified mTORC2 similarly to its known substrate, AKT. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decrease. This treatment also damages colocalization of actin filaments and FLNA. Moreover, treatments with PP242 or RICTOR siRNA can alleviate phosphorylated MYH9 level at the regulatory residue (Ser1943). The significant alteration of the colocalization of actin filaments and MYH9 can also be observed. Altogether, the results support FLNA and MYH9 as downstream effectors of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA and mTORC2-MYH9 signaling pathways plays important roles in motility and invasion of GBM cells.

Ultimately, the discovery of FLNA and MYH9 as novel substrates of mTORC2 will prominently expand the area of studies in both normal, and cancer cells including other disease-causing cells especially the ones that are highly motile.