Role of AMPK in regulating morphogenetic processes during cancer and embryonic development

Abstract

Title: Role of AMPK in regulating morphogenetic processes during cancer and development

Abstract: AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, integrating metabolic status with transcriptional control. Although traditionally viewed as a tumour-suppressor, recent evidence from our lab and others reveals its paradoxical yet key role in the morphogenetic programme of epithelial–mesenchymal transition (EMT) and promoting cancer metastasis. In this study, we investigate how AMPK modulates the EMT master regulator TWIST1 in driving cancer cell plasticity and explore whether the link between energy sensing and morphogenetic processes represents a conserved mechanism shared with developmental context. We employed a multi-pronged research approach, involving mass spectrometry-based proteomics, molecular assays in cancer cell lines, and a developmental model, Drosophila, to dissect the functions of AMPK.

TMT-based quantitative proteomics identified five key TWIST1 interactors—EIF4E, hnRNPA2/B1, CSTB, ANXA2, and SERBP1—whose association was bidirectionally modulated by AMPK activity, revealing AMPK-dependent reprogramming of the TWIST1 interactome across RNA processing, translation, proteostasis, and cytoskeletal organization. Parallel phospho-proteomic analysis uncovered a novel AMPK-dependent phosphorylation site on TWIST1. Stable cell lines expressing phospho-deficient and phospho-mimetic mutants demonstrated that this Serine phosphorylation enhances nuclear localization, stability, and EMT-gene expression, promoting invasive 3D morphology and stem-like traits. The phospho-dead mutant exhibited altered subcellular localization, reduced stability, diminished EMT marker expression, and impaired spheroid formation, AMPK-mediated phosphorylation is critical for maintaining the transcriptional activity and functional integrity of TWIST1. Furthermore, TWIST1 overexpression elevated AMPK activity suggesting a positive feedback loop that reinforces EMT. Together, these findings define AMPK- dependent serine phosphorylation as a novel stabilizing modification of TWIST1 and establish the AMPK–TWIST1 axis as a conserved signaling module linking cellular stress sensing to morphogenesis and metastasis.

In Drosophila embryos, confocal imaging revealed for the first time that phosphorylated AMPK (pAMPK, active) dynamically localizes to spindle poles and metaphase plates, implicating its role in mitotic and cytoskeletal regulation during the syncytial and cellularization stages—key morphogenetic phases of early embryonic development. AMPK depletion disrupted actin organization, epithelial polarity, and embryonic viability, underscoring its essential role in maintaining epithelial integrity and morphogenesis. Thus, by integrating stress signals with cytoskeletal and transcriptional control, AMPK emerges as a central morphogenetic regulator with conserved roles in embryonic development and cancer progression.