Integrated proteomics and phosphoproteomics analyses of esophageal cancer cells with different invasive abilities

Esophageal squamous cell carcinoma (ESCC) is one of the aggressive and lethal malignancies with an extremely poor prognosis. It is necessary to explore the molecular mechanisms of ESCC invasion. We utilized high-throughput mass spectrometry to analyze the proteomes and phosphorylation profiles of two ESCC cell lines with differing invasion capacities (HK vs TE10). Differentially expressed proteins and phosphorites were identified, followed by comprehensive bioinformatics analyses encompassing function and pathway enrichment, protein-protein interaction (PPI) network analysis, hub gene identification, co-expression analysis, kinase-substrate prediction, and drug-target network analysis. CCK-8 assay, transwell examination, wound-healing assay, and western blot was used to validate the effects of fostamatinib on ESCC cells proliferation, invasion, migration, and LYN expression. The Q4 cluster of differentially phosphorylated proteins was primarily associated with functions and pathways relevant to tumor metastasis. Phosphorylated hub proteins including ARHGAP35, CTNNA1, and SHC1 were identified through the analysis of PPI network, and their respective regulated kinases were predicted. Among the predicted kinases, LYN was validated to be associated with lymph node metastasis (N0 vs. N1–3) and prognosis in ESCC patients at mRNA levels using TGGA data and protein levels in ESCC tissues (p