NUAK1 and AMPK control lipid metabolism, mitochondrial dynamics, and myosin-dependent cell migration in mesenchymal colon cancer cells

Abstract A consensus molecular subtype classification has identified four groups (CMS1-4) in colon cancer (CC), with CMS4 associated with the poorest prognosis, largely due to its high metastatic potential. Although the cellular and molecular features of CMS4 tumors have been increasingly characterized, the signaling networks that sustain their mesenchymal and invasive phenotype remain incompletely understood. Here, using the prototypical CMS4 MDST8 cell line under adherent and non-adherent conditions, we investigated the interplay between actomyosin contractility, lipid metabolism, and mitochondrial dynamics. We found that adherent MDST8 cells exhibited a low pAMPK/AMPK ratio, associated with reduced fatty-acid oxidation (FAO), enhanced lipogenesis, and a highly interconnected mitochondrial network. Loss of adherence was accompanied by activation of the NUAK1 (an AMPK-related kinase)-MLC2 (myosin light chain 2) phosphorylation cascade, which was independent of AMPK. Notably, both AMPK and NUAK1, two members of the same kinase family, were activated by LKB1 following cell detachment. Unexpectedly, non-adherent MDST8 cells simultaneously activated FAO and fatty-acid synthesis, two pathways generally considered antagonistic. Detachment from the culture support also induced mitochondrial fission, likely driven by DRP1 phosphorylation at Ser 616 , and we propose that this remodeling of mitochondrial architecture may underlie the concomitant activation of FAO and lipogenesis. Furthermore, we demonstrated that siRNA-mediated knockdown or pharmacological inhibition of DRP1, NUAK1, or AMPK impaired MDST8 cell migration and invasion, underscoring the functional relevance of the signaling cascade activated upon cellular detachment. Finally, analysis of a CC patient cohort revealed that expression of key components of this pathway correlates with clinical outcome, suggesting potential prognostic value. Altogether, our findings uncover a coordinated signaling network linking contractility, lipid metabolism, and mitochondrial dynamics in CMS4 colon cancer cells, and identify actionable molecular targets that may help limit mesenchymal cell dissemination and metastasis.