Chen Yang, Zhang Kun, Wang Xiaosu, Li Qiaoneng, Wu Qingfeng, Ning Xiaoxuan
Department of Geriatrics, Xijing Hospital, Fourth Military Medical University.
College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China.
Anticancer Drugs. 2018 Jan;29(1):19-28. doi: 10.1097/CAD.0000000000000556.
Our previous results showed that calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) inhibits the proliferation and tumorigenicity of gastric cancer; however, the exact mechanism remains unclear, especially from the aspect of cell cycle. The subcellular localization of CacyBP/SIP, Siah-1, and Skp1 in SGC7901 gastric cancer cells was assessed by immunofluorescence after cell cycle synchronization. Levels of CacyBP/SIP, Siah-1, Skp1, β-catenin, and p-ERK1/2 were analyzed by western blotting. CacyBP/SIP phosphorylation (p-CacyBP/SIP) and the combining capacity of Siah-1 and Skp1 with CacyBP/SIP in nucleoprotein were determined by immunoprecipitation. CacyBP/SIP, Siah-1, and Skp1 were mainly in the cytoplasm in the G1 phase, but translocated to the nucleus during G2. Their expression in total protein was not altered, but elevated in the G2 phase in nucleoprotein. The CacyBP/SIP nucleus translocation of cells transfected with mutant CacyBP/SIP that does not bind S100 (CacyBP-ΔS100) was significantly increased compared with wild-type CacyBP/SIP. In the G2 phase, p-CacyBP/SIP expression and the combining capacity of Siah-1 and Skp1 with CacyBP/SIP were all increased, whereas levels of β-catenin and p-ERK1/2 reduced, compared with the G1 phase. CacyBP/SIP or CacyBP-ΔS100 overexpression was correlated with constitutively low β-catenin expression and affected its level through cell cycle. CacyBP/SIP overexpression led to retarded proliferation, G1 arrest, and β-catenin reduction, which could be abolished by lithium chloride, β-catenin activator, and further enhanced by the Wnt inhibitor XAV-939. In addition, CacyBP-ΔS100 further suppressed cell proliferation and induced G1 arrest compared with CacyBP/SIP. In conclusion, CacyBP/SIP nuclear localization, dependent on S100 protein, suppresses gastric cancer tumorigenesis through β-catenin degradation and the dephosphorylation of ERK1/2 during the G2 phase.
我们之前的研究结果表明，钙周期蛋白结合蛋白/与Siah-1相互作用蛋白（CacyBP/SIP）可抑制胃癌的增殖和致瘤性；然而，确切机制仍不清楚，尤其是从细胞周期方面。通过细胞周期同步化后免疫荧光法评估CacyBP/SIP、Siah-1和Skp1在SGC7901胃癌细胞中的亚细胞定位。通过蛋白质印迹法分析CacyBP/SIP、Siah-1、Skp1、β-连环蛋白和p-ERK1/2的水平。通过免疫沉淀法测定CacyBP/SIP磷酸化（p-CacyBP/SIP）以及Siah-1和Skp1与核蛋白中CacyBP/SIP的结合能力。CacyBP/SIP、Siah-1和Skp1在G1期主要位于细胞质中，但在G2期转位至细胞核。它们在总蛋白中的表达未改变，但在G2期核蛋白中升高。与野生型CacyBP/SIP相比，转染不结合S100的突变型CacyBP/SIP（CacyBP-ΔS100）的细胞中CacyBP/SIP核转位显著增加。在G2期，与G1期相比，p-CacyBP/SIP表达以及Siah-1和Skp1与CacyBP/SIP的结合能力均增加，而β-连环蛋白和p-ERK1/2水平降低。CacyBP/SIP或CacyBP-ΔS100过表达与组成性低水平β-连环蛋白表达相关，并通过细胞周期影响其水平。CacyBP/SIP过表达导致增殖受阻、G1期停滞和β-连环蛋白减少，这可被β-连环蛋白激活剂氯化锂消除，并被Wnt抑制剂XAV-939进一步增强。此外，与CacyBP/SIP相比，CacyBP-ΔS100进一步抑制细胞增殖并诱导G1期停滞。总之，依赖于S100蛋白的CacyBP/SIP核定位通过在G2期降解β-连环蛋白和使ERK1/2去磷酸化来抑制胃癌的发生。
