Abstract
The transition of primary tumor cells to a metastatic state is a complex process governed by dynamic signaling events, predominantly regulated by protein phosphorylation. While the genomic landscape of metastasis has been extensively mapped, the functional signaling networks—specifically the kinase-substrate interactomes—remain poorly understood. In this study, we employed a high-resolution, quantitative phosphoproteomics approach using Tandem Mass Tag (TMT) labeling and TiO2-based enrichment to profile the phosphoproteome of highly metastatic versus non-metastatic cancer cell models. Our analysis identified over 12,000 unique phosphorylation sites, providing a deep architectural view of the metastatic signaling landscape. By integrating these data with kinase activity enrichment analysis (KAEA) and substrate prediction algorithms, we reconstructed a comprehensive kinase-substrate network that reveals significant rewiring in metastatic cells. Specifically, we identified a novel signaling axis involving the dysregulation of DCLK1 and GSK3A, which appears to orchestrate epithelial-mesenchymal transition (EMT) and invasive behavior. Furthermore, our results highlight the role of extracellular vesicle-mediated kinase transport in modulating the pre-metastatic niche. Comparison with existing datasets confirmed that these rewired networks are frequently associated with somatic mutations that alter protein recruitment to phosphotyrosine sites. These findings not only provide a high-resolution map of the signaling circuits driving tumor progression but also identify several druggable kinase signatures that could serve as therapeutic targets to circumvent chemoresistance and metastatic spread. This work underscores the necessity of moving beyond static genomic blueprints to functional, dynamic proteomic profiles for the next generation of precision oncology.