背景和目的：食管鳞状细胞癌（esophageal squamous-cell carcinoma,ESCC），简称食管鳞癌，是我国最常见的消化道恶性肿瘤之一，发病率和死亡率均位居前列，且恶性程度高，治疗手段有限，患者预后不良。在多项大规模基因组测序研究中，NOTCH1被鉴定为高频突变基因，并且多为功能失活突变。进一步研究发现，NOTCH1突变在正常衰老的食管上皮驱动克隆扩增，而在食管鳞癌中则抑制肿瘤生长，这种双重作用提示Notch信号具有复杂的调控作用。虽然NOTCH1在食管鳞癌患者中的突变频率约为20%，仅次于TP53，但在更多的NOTCH1未发生突变的食管鳞癌患者中，肿瘤细胞仍然具有功能性的Notch信号。NOTCH1作为Notch信号通路的关键受体，与配体结合后NOTCH1胞内结构域（NOTCH1 intracellular domain, NICD1）向细胞核转位，与RBPJ转录复合体结合，促进下游靶基因的转录。但是，Notch信号通路的激活在食管鳞癌中发挥的具体功能和分子机制尚不明确。

方法：本研究利用已发表的食管鳞癌单细胞转录组数据集，对肿瘤上皮细胞进行Notch信号通路评分及通路富集分析，鉴定Notch信号通路上调可能发挥的潜在功能。利用包含312例食管鳞癌患者临床肿瘤样本的组织芯片，通过多重荧光免疫组化染色检测Notch信号通路激活水平与肿瘤微血管密度之间的相关性。通过体外人脐静脉内皮细胞迁移实验和小管形成实验，以及体内Matrilgel plug实验、小鼠皮下移植瘤模型和化学致癌剂4-NQO诱导的小鼠原发性食管鳞癌模型，验证肿瘤细胞中Notch信号通路激活对肿瘤血管生成的影响。通过蛋白质免疫印迹实验测定肿瘤细胞中促血管生成因子的表达，酶联免疫吸附实验测定肿瘤细胞上清液中促血管生成因子的含量。联合分析CUT&Tag和转录组测序数据，筛选食管鳞癌中Notch信号通路激活的直接靶基因，并通过ChIP-qPCR和双荧光素酶报告基因实验进行转录调控关系的验证。体内治疗实验应用Notch信号下游靶基因的抑制剂，检测其对肿瘤生长和肿瘤中的微血管密度的作用，以及与化疗联合治疗的抗肿瘤疗效。

结果：我们发现食管鳞癌肿瘤上皮细胞中的Notch信号通路评分和血管生成通路的评分显著正相关。在食管鳞癌临床肿瘤样本中，Notch信号激活的水平与肿瘤微血管密度、更进展的TNM分期以及较短的患者生存期显著相关。在食管鳞癌细胞系中敲除NOTCH1，在体外可以显著抑制人脐静脉内皮细胞的迁移以及小管形成的能力，并在体内抑制Matrigel plug组织和小鼠皮下移植瘤组织中的微血管密度。与Notch1^+/+小鼠相比，Notch1^-/-小鼠的原发性食管鳞癌肿瘤组织中的微血管密度也显著降低。分子机制上，Notch信号通路的激活，通过NICD1-RBPJ复合体直接促进靶基因USP5的转录。USP5通过其去泛素化酶的活性，抑制了STAT3的泛素化修饰和蛋白酶体的降解，进而促进STAT3介导的促血管生成因子的表达和分泌。USP5在Notch信号通路促进的肿瘤血管生成中至关重要，体内使用USP5抑制剂EOAI3402142显著抑制肿瘤生长和血管生成，并与化疗联合治疗时取得更强的抗肿瘤疗效。

结论：在食管鳞癌中，Notch信号通过NOTCH1-USP5-STAT3轴促进肿瘤血管生成。同时，靶向抑制Notch信号下游USP5的效应，可以抑制肿瘤生长和血管生成，提示Notch信号通路可作为食管鳞癌潜在的治疗靶点。 

Background and Objective: Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignant gastrointestinal tumors in China, characterized by high incidence, mortality, and malignancy, as well as limited therapeutic options and poor patient prognosis. Large-scale genomic studies have identified NOTCH1 as a frequently mutated gene in ESCC, with most mutations leading to functional inactivation. Further investigations revealed that NOTCH1 mutations drive clonal expansion in normal aging esophageal epithelium but suppress tumor growth in ESCC. This dual role underscores the intricate regulatory complexity of Notch signaling, which is highly context-dependent. Although NOTCH1 mutations occur in approximately 20% of ESCC cases, second only to TP53, the majority of ESCC patients lacking NOTCH1 mutations still retain functional Notch signaling in tumor cells. As a key receptor of the Notch pathway, NOTCH1 binds to ligands, leading to nuclear translocation of the NOTCH1 intracellular domain (NICD1), which forms a complex with RBPJ to promote transcription of downstream targets. However, the precise functional roles and molecular mechanisms of Notch signaling activation in ESCC remain unclear.

Methods: In this study, we utilized a published single-cell transcriptomic datasets of ESCC to perform Notch signaling pathway activity scoring and pathway enrichment analysis on tumor epithelial cells. A tissue microarray comprising 312 clinical ESCC samples was analyzed using multiplexed immunofluorescence staining to assess the correlation between Notch signaling activation and microvascular density. In vitro human umbilical vein endothelial cell (HUVEC) migration and tube formation assays, as well as in vivo Matrigel plug assays, subcutaneous xenograft models, and chemical carcinogen-induced primary ESCC mouse models, were employed to validate the impact of aberrant Notch signaling activation on tumor angiogenesis. Western blotting and enzyme-linked immunosorbent assays (ELISA) were used to measure pro-angiogenic factor expression in tumor cells and their secretion into the supernatant. Integrated analysis of CUT&Tag and transcriptomic sequencing data was conducted to identify direct target genes of the activated Notch signaling pathway in ESCC, followed by experimental validation of transcriptional regulatory relationships through ChIP-qPCR and dual-luciferase reporter assays. In vivo therapeutic experiments evaluated the efficacy of an inhibitor targeting downstream effector of Notch signaling on tumor growth and microvascular density, as well as its antitumor efficacy in combination with chemotherapy.

Results: We observe a significant positive correlation between Notch signaling activity and angiogenesis pathway scores in ESCC tumor epithelial cells. In clinical ESCC samples, Notch signaling activation scores are significantly correlated with tumor microvascular density, advanced TNM stages and short patient survival time. NOTCH1 knockout in ESCC cell lines significantly inhibits HUVEC migration and tube formation in vitro, and reduces microvascular density in Matrigel plugs and subcutaneous xenografts in vivo. Primary ESCC tumors from Notch1-/- mice exhibit markedly lower microvascular density compared to those from Notch1+/+ mice. Mechanistically, Notch signaling activation directly promotes transcription of the target gene USP5 via the NICD1-RBPJ complex. USP5, functioning as a deubiquitinase, stabilizes STAT3 by inhibiting its ubiquitination and proteasomal degradation, thereby enhancing STAT3-mediated expression and secretion of pro-angiogenic factors. USP5 is essential for Notch signaling-driven tumor angiogenesis. In vivo administration of the USP5 inhibitor EOAI3402142 significantly suppresses tumor growth and angiogenesis, and demonstrates enhanced antitumor efficacy when combined with chemotherapy.

Conclusion: Notch signaling promotes ESCC tumor angiogenesis through the NOTCH1–USP5–STAT3 axis. Targeting USP5, the downstream effector of Notch signaling, effectively inhibits tumor growth and angiogenesis, highlighting the Notch signaling as a potential therapeutic target for ESCC.