Abstract
RSK plays a central role in oncogenic signaling, yet its broader regulatory impact in esophageal squamous cell carcinoma (ESCC) remains unclear. We performed integrated phosphoproteomics and kinase-substrate network analysis to elucidate global signaling alterations following RSK inhibition in ESCC cells. In Eca-109 cells, treatment with a moderate concentration of the selective RSK inhibitor SL0101 effectively suppressed proliferation without inducing apoptosis. To elucidate potential compensatory survival mechanisms, we performed integrated proteomic and phosphoproteomic analyses, which revealed that RSK inhibition provokes broad cellular adaptations. Importantly, the magnitude of these effects varied across pathways. Ribosome biogenesis and mitochondrial organization exhibited substantial perturbation primarily at the proteomic level, reflecting system-level dysregulation. Phosphoproteomic analysis revealed that large-magnitude alterations in nuclear transport and mRNA processing, and more subtle, fine-tuned modulation of mitotic fidelity. Kinase activity inference further identified MAPK14, CDK2, SRPK3, AKT1, PLK1, and PIM2 as principal regulators that may regulate compensatory signaling in response to RSK suppression. Taken together, our study reveals that RSK inhibition reprograms kinase networks to enforce stress adaptation, maintaining oncogenic homeostasis despite perturbations. These findings highlight a potential therapeutic window for early intervention strategies, suggesting that combination strategies may enhance the efficacy of RSK-directed therapies in ESCC.