PTEN/β-catenin/Nanog干细胞通路调控鼻咽癌放疗抵抗的实验研究

Radiotherapy is the first choice for the treatment of nasopharyngeal carcinoma (NPC) treatment. NPC often develops recurrence and metastasis because of radiation resistance of cancer stem cells after radiotherapy. PTEN is a prognostic marker in postoperative radiotherapy for head and neck cancer. Wnt/β-catenin pathway emerges as a regulator of cancer stem cell. In our previous research we inhibited the expression of PTEN by siRNA, combined with inhibitor of the Wnt pathway, we proved that PTEN can negatively regulate downstream factors of β-catenin and stem cell associated protein Nanog. We aslo confirmed that NPC cells with low expression of PTEN resist against X rays which requires Wnt pathway. The next step of this project we will validate PTEN/β-catenin /Nanog pathway is involved in the genetic mechanism of NPC stem cell by down-regulating β-catenin, using SP, Aldefluor and tumor sphere formation detection. Furthermore, we will examine the radio-resistance effect of PTEN/β-catenin/Nanog on NPC cells by plate cloning test, comet assay, EdU assay， cell cycle and apoptosis detection. We will investigate the correlation between the pathway and NPC stem cells, radiotherapy prognosis in vivo and clinical samples respectively. This project has important meaning to explore stem cell genetics, radiation tolerance mechanism and find a new treatment method of NPC.

放射治疗是鼻咽癌（NPC）首选治疗方式，肿瘤干细胞放疗抵抗是导致治疗失败的主要原因。PTEN表达同头颈部癌放疗预后相关，Wnt/β-catenin通路参与肿瘤干细胞调节。课题组前期通过干扰PTEN表达，结合Wnt通路抑制剂，证明PTEN可负性调节下游β-catenin和干细胞相关蛋白Nanog，并证实低表达PTEN的NPC细胞可通过调节Wnt通路抵抗X射线，拟下一步结合干扰β-catenin，利用SP和Aldefluor检测以及肿瘤球实验验证PTEN/β-catenin/Nanog通路对NPC干细胞的调节，进一步通过平板克隆实验、EdU实验、彗星实验以及细胞周期和凋亡检测研究PTEN/β-catenin/Nanog通路对NPC放射抵抗的影响，并拟在活体和临床样本内观察该通路同NPC干细胞和放疗预后的相关关系。本项目对于探索NPC干细胞发生机制、放疗抵抗机制以及探索新的治疗方法具有重要意义。

放射治疗是鼻咽癌（NPC）首选治疗方式，肿瘤干细胞放疗抵抗是导致治疗失败的主要原因。本项目首先利用FACS分选NPC细胞株CNE1和CNE2细胞的PTEN-细胞，克隆形成实验结果显示PTEN-细胞较PTEN+细胞更具备放射抵抗性，PTEN-细胞较PTEN+表达更高的干细胞相关蛋白，4Gy X射线处理PTEN-/+细胞，PTEN-较PTEN+细胞表达DNA损伤小。利用siRAN构建低表达PTEN的NPC细胞，降低PTEN表达可提高侧群细胞（SP）比例，PI3K抑制剂LY294002可阻止这一效应，siPTEN细胞表达更高的p-AKT，active-β-catenin和Nanog，LY294002可阻止这一效应。降低PTEN表达可诱导更多肿瘤球形成，LY294002和AKT抑制剂Perifosine可阻止这一效应。降低PTEN表达可增强细胞对放射抵抗，LY294002和Perifosine可阻止这一效应。4Gy X射线处理细胞，降低PTEN表达可减轻细胞DNA损伤，LY294002可阻止这一效应。shRAN构建低表达β-catenin的NPC细胞，干扰β-catenin可降低Nanog表达和SP细胞比例。4Gy X射线处理细胞，干扰β-catenin可降低肿瘤球形成。克隆形成实验显示干扰β-catenin可明显提高PTEN-细胞放射敏感性，PI3K/AKT激活剂IGF-1可逆转其敏感性。干扰PTEN表达可促使β-catenin由胞浆转入胞核，经X射线处理的PTEN-的细胞active-β-catenin表达于细胞核。免疫组化分析NPC病理组织显示PTEN-和p-AKT+/核β-catenin+/Nanog+表达有相关性，生存期分析显示PTEN-/pAKT+/核β-catenin+/Nanog+ NPC病人放疗后预后差。NOD-SCID鼠成瘤实验结果显示Salinomycin可以逆转siPTEN细胞引起的放疗抵抗。本项目得出结论：PTEN-的NPC细胞具有干细胞特性和放射抵抗特性，PTEN通过PI3K/AKT/β-catenin调控NPC干细胞和放射抵抗，此过程伴随着β-catenin由胞浆转入胞核，PTEN-/p-AKT+/核β-catenin+/Nanog+表达的NPC患者放疗预后差。本项目对于探索NPC干细胞发生机制、放疗抵抗机制以及探索新的治疗方法具有重要意义。