NCSTN 通过 Notch1/AKT 依赖性途径激活 β-连环蛋白促进肝癌细胞生长和转移。
NCSTN promotes hepatocellular carcinoma cell growth and metastasis via β-catenin activation in a Notch1/AKT dependent manner.
机构信息
Department of Liver Surgery, Liver Transplantation Division, Laboratory of Liver Surgery, West China Hospital, Sichuan University, Chengdu, 610041, China.
Laboratory of Liver Surgery, West China Hospital, Sichuan University, Chengdu, 610041, China.
出版信息
J Exp Clin Cancer Res. 2020 Jul 6;39(1):128. doi: 10.1186/s13046-020-01638-3.
BACKGROUND
Hepatocellular carcinoma is the third top cause of cancer-related mortalities worldwide. The prognosis of HCC patients remains poor due to rapid progression and high incidence of tumor recurrence. Nicastrin (NCSTN), a core subunit of γ-Secretase, has been reported to play a vital role in tumor progression. However, no study till now has revealed its role in HCC.
METHODS
The expression of NCSTN was evaluated by immunohistochemical staining, Western blot, and quantitative real-time PCR. Cell counting kit-8, colony formation and cell cycle assays were used for evaluating cell growth in vitro. Transwell and wound-healing assays were used for evaluating cell migration and invasion capacity. Immunofluorescence, subcellular protein fractionation and co-immunoprecipitation were used for location analysis of β-catenin. The in vivo functions of NCSTN were illustrated by xenograft tumor models.
RESULTS
NCSTN was dramatically overexpressed in HCC compared to normal liver tissues. Elevated NCSTN expression level was significantly correlated to worse overall and recurrence-free survival of HCC patients. Enhanced NCSTN expression promoted HCC cell growth, migration and invasion in vitro and in vivo. Mechanistic investigations showed that NCSTN induced epithelial-mesenchymal transition (EMT) process via upregulation of Zeb1. Subsequently, we revealed that NCSTN facilitated nuclear translocation of β-catenin, a positive transcriptional regulator of Zeb1. Using Notch and AKT inhibitors, we revealed that NCSTN promoted β-catenin activation through Notch1 and AKT signaling pathway. NCSTN increased AKT and GSK-3β phosphorylation by cleavage of Notch1, which decreased GSK-3β/β-catenin complex. The inactivation of GSK-3β inhibited the β-catenin degradation and promoted nuclear translocation of β-catenin to initiate transcription of Zeb1, resulting in malignant phenotype.
CONCLUSIONS
Our results demonstrated that NCSTN promoted HCC cell growth and metastasis via β-catenin-mediated upregulation of Zeb1 in a Notch1/AKT dependent manner, suggesting that NCSTN might serve as a potential prognostic marker and therapeutic target for HCC.
背景
肝细胞癌是全球癌症相关死亡的第三大主要原因。由于 HCC 患者病情进展迅速且肿瘤复发率高，其预后仍然较差。γ-分泌酶的核心亚基尼卡斯特林（NCSTN）已被报道在肿瘤进展中发挥重要作用。然而，迄今为止尚无研究表明其在 HCC 中的作用。
方法
通过免疫组织化学染色、Western blot 和实时定量 PCR 评估 NCSTN 的表达。细胞计数试剂盒-8、集落形成和细胞周期测定用于评估体外细胞生长。Transwell 和划痕愈合测定用于评估细胞迁移和侵袭能力。免疫荧光、亚细胞蛋白分级和共免疫沉淀用于β-连环蛋白的定位分析。通过异种移植肿瘤模型说明了 NCSTN 的体内功能。
结果
与正常肝组织相比，NCSTN 在 HCC 中明显过表达。NCSTN 表达水平升高与 HCC 患者总生存期和无复发生存期较差显著相关。增强的 NCSTN 表达促进了 HCC 细胞在体外和体内的生长、迁移和侵袭。机制研究表明，NCSTN 通过上调 Zeb1 诱导上皮-间充质转化（EMT）过程。随后，我们揭示了 NCSTN 通过 Notch1 促进β-连环蛋白的核易位，Zeb1 的一个阳性转录调节剂。使用 Notch 和 AKT 抑制剂，我们揭示了 NCSTN 通过 Notch1 和 AKT 信号通路促进β-连环蛋白的激活。NCSTN 通过 Notch1 的切割增加 AKT 和 GSK-3β 的磷酸化，从而减少 GSK-3β/β-连环蛋白复合物。GSK-3β 的失活抑制了β-连环蛋白的降解并促进了β-连环蛋白的核易位，从而启动 Zeb1 的转录，导致恶性表型。
结论
我们的研究结果表明，NCSTN 通过 Notch1/AKT 依赖性途径促进 HCC 细胞的生长和转移，通过β-连环蛋白介导的 Zeb1 上调，提示 NCSTN 可能作为 HCC 的潜在预后标志物和治疗靶点。
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