Dysregulation of TUT4/7 alters AKT1 signaling in epithelial ovarian cancer metastasis
RNA 3' end modifications regulate stability, impacting cellular processes such as proliferation and metastasis. Terminal uridylyltransferases 4/7 (TUT4/7), responsible for untemplated addition of uridine residues to RNAs, play essential RNA regulation roles, altering let-7 family miRNA abundance, which has been linked to AKT1 phosphorylation at T308 and S473, key sites for AKT1 activity. AKT1 activity regulates epithelial ovarian cancer (EOC) metastatic behaviour, but how AKT1 phosphorylation is regulated remains unresolved. We identified decreased AKT1 phosphorylation upon upstream TUT4/7 inhibition, using the small molecule inhibitor LIN28i 1632. To probe the roles of TUT4/7 in EOC metastatic behaviour, we generated CRISPR/Cas9 single- and double-knockouts of TUT4/7 in OVCAR8 EOC cells, and measured abundance of let-7 miRNAs, AKT1 phosphorylation, and AKT1 target phosphorylation. We identified differential changes in let-7 miRNA abundance in single- and double-knockout cells, highlighting distinct regulatory roles for each enzyme. Single knockouts of TUT4/7 increased AKT1 T308 phosphorylation, whereas double knockout cells predominantly elevated phosphorylation at AKT1 S473. TUT4-knockout was linked to increased abundance and phosphorylation of the AKT1 substrate GSK-3a. Metabolomic profiling revealed decreased non-polar amino acids in TUT4-knockout cells, suggesting altered amino acid metabolism linked to AKT signaling. Together, these results implicate TUT4 and AKT1 activity in EOC.
RNA 3' end modifications regulate stability, impacting cellular processes such as proliferation and metastasis. Terminal uridylyltransferases 4/7 (TUT4/7), responsible for untemplated addition of uridine residues to RNAs, play essential RNA regulation roles, altering let-7 family miRNA abundance, which has been linked to AKT1 phosphorylation at T308 and S473, key sites for AKT1 activity. AKT1 activity regulates epithelial ovarian cancer (EOC) metastatic behaviour, but how AKT1 phosphorylation is regulated remains unresolved. We identified decreased AKT1 phosphorylation upon upstream TUT4/7 inhibition, using the small molecule inhibitor LIN28i 1632. To probe the roles of TUT4/7 in EOC metastatic behaviour, we generated CRISPR/Cas9 single- and double-knockouts of TUT4/7 in OVCAR8 EOC cells, and measured abundance of let-7 miRNAs, AKT1 phosphorylation, and AKT1 target phosphorylation. We identified differential changes in let-7 miRNA abundance in single- and double-knockout cells, highlighting distinct regulatory roles for each enzyme. Single knockouts of TUT4/7 increased AKT1 T308 phosphorylation, whereas double knockout cells predominantly elevated phosphorylation at AKT1 S473. TUT4-knockout was linked to increased abundance and phosphorylation of the AKT1 substrate GSK-3a. Metabolomic profiling revealed decreased non-polar amino acids in TUT4-knockout cells, suggesting altered amino acid metabolism linked to AKT signaling. Together, these results implicate TUT4 and AKT1 activity in EOC.