Department of Pathology, Shantou Central Hospital, Affiliated Shantou Hospital of Sun Yat-sen University, Shantou 515041, China; Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Shantou University Medical College, Shantou 515041, China; Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China.
Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Shantou University Medical College, Shantou 515041, China; Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China.
Cell Signal. 2019 Mar;55:8-16. doi: 10.1016/j.cellsig.2018.12.007. Epub 2018 Dec 14.
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.
高表达的脂联素 2（LCN2）通常预示着食管鳞状细胞癌（ESCC）和许多其他癌症的预后不良。我们之前的研究表明，LCN2 通过一个新的正反馈环促进 ESCC 细胞的迁移和侵袭。然而，该环中的关键转录激活蛋白（KTAP）尚未被鉴定。在这项研究中，我们首先通过生物信息学分析预测最有可能的 KTAP。然后，我们通过融合其 5'侧翼区域缺失来评估人 LCN2 基因的转录调控区到双荧光素酶报告基因。我们发现，包含转录因子 7 样 2（TCF7L2）（-273/-209）和早期生长反应 1（EGR1）（-710/-616）结合位点的-720/-200 区域对于 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 作为 KTAP 直接结合到 LCN2 启动子内的其结合位点。从机制上讲，TCF7L2 和 EGR1 的过表达通过 ERK 信号通路增加内源性 LCN2 的表达。重组人 LCN2 蛋白的处理增强了 ERK 通路的激活，以促进内源性 LCN2 的表达，并增加 TCF7L2 和 EGR1 的表达水平。用 MEK 抑制剂 U0126 处理可抑制 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。在放大 LCN2 的诱导并增强迁移和侵袭方面，TCF7L2 和 EGR1 之间观察到协同作用。总之，我们的研究表明，TCF7L2 和 EGR1 是 LCN2 的 KTAP，在正“LCN2→MEK/ERK→LCN2”途径内，促进 ESCC 细胞的迁移和侵袭。基于其临床病理意义，LCN2 及其两个表达调节剂 TCF7L2 和 EGR1 可能成为 ESCC 的治疗靶点。
