Multi-omics analysis of epithelial ovarian cancer metastasis
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.