食管鳞状细胞癌（ESCC）是一种具有极差预后的侵袭性和致命性肿瘤之一。探索ESCC侵袭的分子机制是必要的。我们利用高通量质谱分析了具有不同侵袭能力的两种ESCC细胞系（HK与TE10）的蛋白质组和磷酸化谱。鉴定了差异表达蛋白和磷酸化物质，并进行了全面的生物信息学分析，包括功能和通路富集、蛋白质相互作用（PPI）网络分析、中心基因鉴定、共表达分析、激酶底物预测和药物靶标网络分析。通过CCK-8测定、Transwell检测、划痕愈合实验和免疫印迹验证了fostamatinib对ESCC细胞增殖、侵袭、迁移和LYN表达的影响。不同磷酸化蛋白质的Q4簇主要与肿瘤转移相关的功能和通路有关。通过PPI网络分析鉴定了包括ARHGAP35、CTNNA1和SHC1在内的磷酸化中心蛋白质，并预测了它们各自的调控激酶。在预测的激酶中，LYN经TGGA数据验证与淋巴结转移（N0 vs. N1-3）和ESCC患者预后在mRNA水平上相关（p<0.05），并在ESCC组织中的蛋白水平也得到验证。验证实验确认了fostamatinib抑制ESCC细胞增殖、迁移、侵袭和LYN表达的效果。我们的多组学分析提供了对ESCC侵袭性的更深入的视角，并揭示了具有调控激酶的新的磷酸化中心蛋白质。本研究还表明，fostamatinib可能是治疗ESCC的潜在药物。版权所有 © 2023. Elsevier Inc.出版

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 < 0.05). Validation experiments confirmed the inhibitory effects of fostamatinib on ESCC cells proliferation, migration, invasion, and LYN expression.Our multi-omics analysis offers deeper perspectives on ESCC invasiveness and unveils new phosphorylated hub proteins with their regulatory kinase. This study also suggests that fostamatinib may be a potential agent for treating ESCC.Copyright © 2023. Published by Elsevier Inc.