Notch1通路激活并上调ephrinB1表达在促骨肉瘤细胞“干性”及增加化疗耐药中的作用与机制

Although it is well known that the high malignancy and poor prognosis of osteosarcoma are closely related with chemoresistance, the mechanism remains obscure. Our study group has cultivated several chemoresistant cell lines of osteosarcoma by drug exposure. The phenotype and molecule detection has revealed a capacity enhancement of organoid formation in vitro and tumor formation in vivo, and an evident overexpression of stem-cell-related gene Oct4 and Sox2, accompanied with an activation of Notch signaling pathway. Notch1 inhibitor would reduce the stem-cell-like characteristics of these cell lines, increasing their sensitivity to chemotherapy. Meanwhile, we discovered the Notch could upregulate the expression of ephrinB1. These results prompt that the Notch1 activation stands a significant role in the stem-cell formation and chemoresistance of osteosarcoma, which may have relations with ephrinB1 upregulation. Based on this hypothesis, our research would clarify the downstream regulatory mechanism of Notch1, its effect on ephrinB1-EphB signaling pathway, stem-cell formation and chemoresistance of osteosarcoma. This project aims to provide an theoretical and experimental basis for new strategies and targets for overwhelming the chemoresistance of osteosarcoma.

骨肉瘤恶性程度高、预后差与化疗耐药密切相关，但相关机制不清。本课题组前期通过药物诱导获得了多个骨肉瘤耐药细胞株，对其表型及相关分子检测发现，耐药株的体外悬浮成球及小鼠体内的成瘤能力均显著增强，干细胞相关基因Oct4、Sox2等的表达明显上调，同时伴有Notch1信号通路的激活；用Notch1抑制剂可明显减少上述耐药细胞株的“干性”特征并增强细胞对化疗的敏感性，同时发现Notch1通路可上调ephrinB1的表达。上述结果提示，Notch1通路的激活在促骨肉瘤细胞的“干性”及化疗耐药中发挥重要作用，并可能同Notch1对ephrinB1的表达上调有关。本研究拟在上述工作基础上，系统探索Notch1信号通路及该通路对ephrinB1-EphB信号通路的调控在促骨肉瘤细胞“干性”形成及化疗耐药中的作用及机制，以期能为探索克服骨肉瘤化疗耐药的新策略和新靶点提供理论及实验依据。

尽管骨肉瘤的诊断和治疗取得了重大进展，但化疗耐药的分子机制仍不清楚。本项目提供了强有力的临床和实验证据，证明Notch信号通路的激活有助于骨肉瘤的进展和化疗耐药。首先，临床资料中12例患者的队列研究发现，与相邻正常组织相比，肿瘤中Notch基因上调，Notch1域(NICD1)和Notch靶基因Hes1的肿瘤高表达与化疗不良反应有关。对公开数据集的数据挖掘证实了Notch通路基因的表达与骨肉瘤的不良预后有关。在体外分析的基础上，Notch信号通路促进骨肉瘤的增殖，增强化疗耐药性，促进迁移和侵袭，并上调干细胞样特性。异种移植瘤模型表明，Notch信号通路促进原发肿瘤生长和肺转移，抑制Notch信号通路可有效减小肿瘤大小和防止转移。在机制上，激活的Notch信号通路诱导ephrinB1的表达，并增强肿瘤促进ephrin反向信号通路。总之，这些发现为Notch通路基因作为候选生物标志物预测骨肉瘤患者的预后提供了功能证据，并为使用Notch抑制剂治疗骨肉瘤提供了机制基础。