Notch signaling regulates osteosarcoma proliferation and migration through Erk phosphorylation
Notch signaling regulates osteosarcoma proliferation and migration through Erk phosphorylation
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DOI：
10.1016/j.tice.2019.07.002
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发表时间：
2019-08-01
期刊：
影响因子：
2.6
通讯作者：
Wang, Dong
中科院分区：
文献类型：
--
作者：
Qin, Jie;Wang, Rui;Wang, Dong
我们使用具有高转移潜力的小鼠自发骨肉瘤细胞系K7M2细胞系来研究Notch信号在骨肉瘤生物学表现中的作用，了解其调节细胞增殖和迁移的潜在机制，并通过发现新的治疗靶点来改善骨肉瘤患者的预后。首先，通过免疫染色和γ-分泌酶抑制剂测定K7M2中Notch的表达N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰]-S-苯基甘氨酸叔丁酯 (DAPT) 用于抑制 Notch 胞内结构域 (NICD) 的蛋白水解切割，从而抑制 Notch 激活。通过使用Sulforhodamine B测定、集落形成单位测定、Brdu和Ki67染色以及细胞凋亡和细胞周期阶段的流式细胞术测定，发现DAPT以剂量依赖性方式抑制K7M2增殖。通过使用伤口愈合和跨孔迁移测定，发现 DAPT 也以剂量依赖性方式抑制 K7M2 迁移。通过结合显微拉曼光谱和 K 均值聚类分析，我们发现 DAPT 抑制大多数 K7M2 细胞结构中多种重要的细胞代谢相关成分。然后，发现DAPT以浓度依赖性方式抑制Notch1ICD表达，并且通过Western blotting发现该表达与Phospho-Erk1/2 (p-Erk)直接相关。为了证实这一发现，我们使用Notch信号配体Jagged1激活Notch信号通路，进而上调p-Erk，导致K7M2的增殖和迁移增加。使用 Erk 通路抑制剂 U0126，我们发现 p-Erk 下调，K7M2 的增殖和迁移也随之减少。最后，我们构建了K7M2小鼠胫骨旁肿瘤模型和肺转移模型。我们发现DAPT在体内抑制p-Erk，有效控制肿瘤生长，减少血管生成，减少肺部转移，并提高总体生存率。综上所述，Notch信号在骨肉瘤中发挥癌基因作用并通过p-Erk促进转移。 DAPT通过抑制Erk磷酸化，在体内外有效抑制骨肉瘤的增殖和转移。因此，抑制Notch激活导致Erk通路磷酸化下调可作为临床治疗中改善骨肉瘤预后的潜在治疗靶点。
We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.