NUAK1 directly induces Akt signaling and substrate specificity, promoting cancer cell survival

Background: NUAK1 is a serine/threonine kinase member of the AMPKα-family, whose high expression is associated with poor prognosis. However, NUAK1 regulation and functions in cancer remain poorly characterized. Here, we investigated NUAK1’s role in cancer signaling. Materials and Methods: We performed a bioinformatic analysis of TCGA to study the correlation between NUAK1 expression and the expression of EGFR and Akt phosphorylation in several cancers. We mainly performed the studies on breast and colon cancer cell lines. Pharmacological or shRNA-dependent inhibition of NUAK1 demonstrated its role in the EGFR and insulin-dependent signaling. In vitro kinase assays were performed to demonstrate the direct phosphorylation of Akt by NUAK1, and proximity ligation assays demonstrated the association of NUAK1 with Akt in cancer cells. Cell fractionation and immunofluorescence studies analyzed NUAK1 subcellular localization, and qPCR studies demonstrated its role in the Akt/ FOXO1/3a axis. We used 2D and 3D cultures for cell survival studies and monitored cell death using commercial kits. Results: Based on public TCGA data, we reported that NUAK1 expression correlates with EGFR expression and the Akt phosphorylation at Ser-473 in several cancers. Using pharmacological inhibition and shRNA-dependent silencing, we found that NUAK1 activates the Akt signaling, regulating FOXO1/3a and GSK3β phosphorylation but not TSC2 phosphorylation. Mechanistically, NUAK1 interacts with Akt and directly phosphorylates it at Ser-473. Comparing NUAK1 and mTOR inhibition revealed an Akt-dynamic activation and -substrate specificity depending on its phosphorylation by NUAK1 or mTORC2. The Akt-substrate specificity could be directly associated with NUAK1 co-localization with early endosomes. Functionally, the NUAK1/Akt/FOXO1/3a axis reduced p21CIP1 and p27KIP1 expression but induced FoxM1 expression. Additionally, our study identified that NUAK1 promotes cancer cell survival in a growth factor-dependent manner, and its inhibition potentiates the effect of MK-2206, an Akt inhibitor. Conclusion: We demonstrated that NUAK1, in contrast to other AMPK-related members, regulates cancer signaling via direct Akt phosphorylation. Thus, targeting NUAK1, either alone or combined with Akt inhibitors, may be effective in cancers with hyperactivated Akt signaling. Funding: ANID/Fondecyt 1191172 and 1201215 No conflict of interest.