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.

Ras homolog enriched in brain (RHEB) is a member of the Ras superfamily of small GTPases that are responsible for the activation of numerous diverse cellular pathways in the cell. Rheb directly activates mTORC1, a crucial signaling pathway that stimulates protein synthesis and cell proliferation. Aberrant Rheb/mTORC1 signaling has been linked to many diseases including cancer. Unfortunately, mTORC1 inhibitors often display only partial antitumor activity, with tumor regrowth upon discontinuation of the drugs. Some reports suggest Rheb may have mTORC1-indpendent functions, but studies to identify additional Rheb effector proteins are lacking.

The aim of this dissertation is to identify new effector proteins of Rheb, and characterize the signaling pathways affected by Rheb interaction with these proteins. To accomplish this, I employ various biochemical techniques. The main approach in identifying new effector proteins is to look for proteins that do not interact with Rheb effector domain mutants, show similar co-localization with Rheb, and whose function is altered upon Rheb interaction.

In this dissertation I present results identifying four different effector proteins of Rheb, and explore the biological relevance of these interactions. First I confirm Rheb interaction with mTOR and TSC. Second, I show a novel interaction between Rheb and CAD, the protein responsible for carrying out the first three enzymatic steps in de novo pyrimidine biosynthesis. In this section I present results demonstrating Rheb stimulates pyrimidine biosynthesis through CAD. Third, I show Rheb interaction with AMPK, a kinase complex that shuts down cell growth and proliferation. Fourth, I show RHEB interaction with BRAF that results in the inhibition of the MAPK pathway. In this section I identify a RHEB mutation from cancer genome database analysis, and demonstrate its ability to transform normal cells into cancer cells through stimulation of the RAF/MEK/ERK signaling pathway.

In conclusion, this dissertation identifies new effector proteins of Rheb and significantly expands our current knowledge of Rheb signaling.

Mesoporous silica nanoparticles (MSNs) are attractive drug delivery vehicle candidates due to their biocompatibility, stability, high surface area and efficient cellular uptake. In this dissertation, I discuss three aspects of MSNs' cellular behavior. First, MSNs are targeted to primary and metastatic cancer cell lines, then their exocytosis from cancer cells is studied, and finally they are used to recover intracellular proteins. Targeting of MSNs to primary cancer cells is achieved by conjugating transferrin on the surface of the mesoporous framework, which resulted in enhancement of nanoparticle uptake and drug delivery efficacy in cells that overexpress the transferrin receptor. Similarly, RGD peptides are used to target metastatic cancer cell lines that over-express integrin &alpha&nu&beta₃. A circular RGD peptide is bound to the surface of MSNs and the endocytosis and cell killing efficacy of camptothecin loaded nanoparticles is significantly improved in cells that express the target receptor. Besides targeting, I studied the ultimate fate of phosphonate coated mesoporous silica nanoparticles inside cells. I discovered that the nanoparticles are exocytosed from cells through lysosomal exocytosis. The nanoparticles are exocytosed in intact form and the time that they remain inside the cells is affected by the surface properties of the nanoparticles and the type of cells. Cells that have a high rate of lysosomal exocytosis excrete the nanoparticles rapidly, which makes them more resistant to drug loaded nanoparticles because the amount of drug that is released inside the cell is limited. When the exocytosis of MSNs is inhibited, the cell killing efficacy of nanoparticles loaded with camptothecin is enhanced. The discovery that MSNs are exocytosed by cells led to a study to determine if proteins could be recovered from the exocytosed nanoparticles. The procedure to isolate exocytosed zinc-doped iron core MSNs and identify the proteins bound to them was developed. This serves as a foundation to use MSNs as protein harvesting tools and investigate protein expression in cancer cells.

mTOR complex 1 integrates the input from several upstream pathways, including growth factors, insulin and amino acids. It can also sense the nutrient, oxygen and energy status of the cell. The mTOR pathway is often dysregulated in human diseases, specifically cancer. This central integration center is often modulated in order to promote uncontrollable cell growth, regardless of extracellular input. The protein known as Raptor (Regulatory Associated Protein of mTOR) is largely responsible for substrate protein recognition. Not much is known about how Raptor recruits mTOR substrate proteins. This study seeks to better understand the specific contribution of structural regions of the Raptor protein in recognizing and binding mTOR substrate proteins S6K1 and 4E-BP1. This study shows that the conserved N-terminal portion of the Raptor protein is involved in these interactions.

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Background

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Conclusions

From the clinical editor