High level expression of lipocalin 2 (LCN2) usually indicates poor prognosis in esophageal squamous cell carcinoma (ESCC) and many other cancers. Our previous study showed LCN2 promotes migration and invasion of ESCC cells through a novel positive feedback loop. However, the key transcription activation protein (KTAP) in the loop had not yet been identified. In this study, we first predicted the most probable KTAPs by bioinformatic analysis. We then assessed the transcription regulatory regions in the human LCN2 gene by fusing deletions of its 5′-flanking region to a dual-luciferase reporter. We found that the region −720/−200 containing transcription factor 7-like 2 (TCF7L2) (−273/−209) and early growth response 1 (EGR1) (−710/−616) binding sites is crucial for LCN2 promoter activity. Chromatin immunoprecipitation (ChIP) experiments demonstrated that TCF7L2 and EGR1 bound directly to their binding sites within the LCN2 promoter as KTAPs. Mechanistically, overexpression of TCF7L2 and EGR1 increased endogenous LCN2 expression via the ERK signaling pathway. Treatment with recombinant human LCN2 protein enhanced activation of the ERK pathway to facilitate endogenous LCN2 expression, as well as increase the expression level of TCF7L2 and EGR1. Treatment with the MEK inhibitor U0126 inhibited the activation by TCF7L2 or EGR1 overexpression. Moreover, overexpression of TCF7L2 or EGR1 accelerated the migration and invasion of ESCC cells. A synergistic effect was observed between TCF7L2 and EGR1 in amplifying the induction of LCN2 and enhancing migration and invasion. Taken together, our study indicates that TCF7L2 and EGR1 are the KTAPs of LCN2, within a positive “LCN2 → MEK/ERK → LCN2” path, to promote the migration and invasion of ESCC cells. Based on their clinicopathological significance, LCN2 and its two expression regulators TCF7L2 and ERG1 might be therapeutic targets for ESCC.

lipocalin 2（LCN2）的高水平表达通常表明在食管鳞状细胞癌（ESCC）和许多其他癌症中预后较差。我们以前的研究表明，LCN2通过新颖的正反馈回路促进ESCC细胞的迁移和侵袭。但是，尚未发现环中的关键转录激活蛋白（KTAP）。在这项研究中，我们首先通过生物信息学分析预测了最可能的KTAP。然后，我们通过将其5'侧翼区的缺失融合到双荧光素酶报道基因中来评估人LCN2基因中的转录调控区。我们发现，包含转录因子7-like 2（TCF7L2）（-273 / -209）和早期生长反应1（EGR1）（-710 / -616）结合位点的区域-720 / -200对于LCN2启动子活性至关重要。染色质免疫沉淀（ChIP）实验表明，TCF7L2和EGR1以KTAPs的形式直接结合在LCN2启动子内的结合位点上。从机制上讲，TCF7L2和EGR1的过表达通过ERK信号通路增加了内源性LCN2的表达。用重组人LCN2蛋白处理可增强ERK途径的活化，以促进内源性LCN2表达，并增加TCF7L2和EGR1的表达水平。用MEK抑制剂U0126进行的治疗抑制了TCF7L2或EGR1过表达的激活。此外，TCF7L2或EGR1的过表达加速了ESCC细胞的迁移和侵袭。在TCF7L2和EGR1之间观察到协同效应，可放大LCN2的诱导并增强迁移和侵袭。在一起 我们的研究表明，TCF7L2和EGR1是LCN2的KTAP，在正向“ LCN2→MEK / ERK→LCN2”路径内，以促进ESCC细胞的迁移和侵袭。基于其临床病理学意义，LCN2及其两个表达调节剂TCF7L2和ERG1可能是ESCC的治疗靶标。