Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Heinemann, Ilka U.

Abstract

Epithelial ovarian cancer (EOC) is a high-risk cancer with heterogeneous tumors. The high incidence of EOC metastasis from primary tumors to nearby tissues and organs is a major driver of EOC lethality. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT1) pathway regulates cell survival and is over-activated in most human cancers, including EOC. Growth factor stimulation activates AKT1 by phosphorylation at T308 and S473. In my cell-based metastasis model, adherent cells model primary tumors, spheroids represent metastatic spread, and re-adherent spheroid cells represent secondary tumors. In spheroids, I show reduced S473 and T308 phosphorylation of AKT, along with significantly increased levels of five let-7 family miRNAs. Terminal nucleotidyltransferases TUT4 and TUT7 (TUT4/7) regulate let-7 miRNA stability by 3′ end uridylation. I found eight let-7 family miRNAs were increased in abundance in TUT4/7-deleted HEK 293T cells, showing that TUT4/7 indirectly control AKT signaling and phosphorylation. In growth factor-stimulated HEK 293T cells supplemented with let-7a, I found increased AKT1 phosphorylation at T308, decreased S473 phosphorylation, and high downstream AKT1 substrate GSK-3β phosphorylation. Next, I investigated cellular signaling dynamics in each step toward EOC metastasis by TMT-proteomics and -phosphoproteomics. These analyses showed that the dormant status of EOC spheroids is due to reduced Aurora kinase B (AURKB) abundance and downstream substrate phosphorylation, resulting in cell cycle arrest. Phosphoproteome analysis further revealed stimulation of Rho-associated kinase 1 (ROCK1) in spheroid cells, controlling cytoskeletal organization. Application of the ROCK1-specific inhibitor Y-27632 to spheroids increased the rate of re-adherence and spheroid density. Finally, I combined proteomics data with metabolomic investigation of the tricarboxylic acid (TCA) cycle, revealing upregulation of TCA cycle enzymes and activity in spheroids, along with reduced serine biosynthesis and consumption. Lastly, I show spheroids have increased oxidative phosphorylation and electron transport chain proteins. These comprehensive data highlight the previously unexplored relationship between AKT phosphorylation and let-7 miRNAs, provide insights into the systemic changes accompanying EOC metastasis, and highlight the high-energy potential of cells in spheroid culture as priming for re-adherence, advancing our understanding of EOC metastasis.

Summary for Lay Audience

Epithelial ovarian cancer (EOC) can be difficult to target with current chemotherapies. A major problem in treating EOC is disease spread. The ovaries are near other organs in the abdomen, meaning cells from the original tumour can split off and spread to those organs, including the colon and diaphragm, in a process called metastasis. To model metastasis, we grow cells in standard conditions or on special plates to form spheroids, which reflect the spreading of cells from the primary tumour into the abdominal fluid. Returning spheroids to normal growth conditions causes re-adherence, modelling secondary tumours on other organs. Thus, this model includes three growth conditions: adherent/primary tumour-like, spheroid/metastasis-like, and re-adherent/secondary tumour-like. One protein that controls EOC metastasis is AKT (protein kinase B). AKT has two “ON-OFF switches”, threonine 308 (T308) and serine 473 (S473). When either of these sites is activated in an event called phosphorylation, AKT is turned on part way, but only with both phosphorylations is AKT completely on. In the first half of this thesis, I focus on understanding how AKT phosphorylation can be turned on or off by a molecule called let-7 and show that let-7 prevents AKT from being turned completely on by preventing S473 phosphorylation. In the second half of this thesis, I shift focus to investigate changes happening in cells at a global level. Using a method called proteomics, the amounts of thousands of proteins present in adherent, spheroid, and re-adherent cells were measured to identify which proteins control the shift between these states. Lastly, building off the proteomics, I use a method called metabolomics to investigate the functional changes we found in proteomics. Proteomics and metabolomics are similar to food consumption: when eating, certain proteins are turned on or off to aid in breaking down the food. In proteomics, we investigate the proteins that are breaking down the food, while in metabolomics, we look at the food itself and how the food is being used. Together, these data offer insight to how EOC metastasis occurs and provide potential avenues for future treatments for EOC.

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