EGFRvⅢ/Akt/PKcs通路在低剂量超微分割照射克服胶质瘤干细胞放疗抵抗的作用及机制

Radiotherapy is the most effective nonsurgical treatment modality yet recurrence is essentially universal. Glioblastoma stem cells (GSCs), which was able to initiate or reconstitue tumor, is highly resistant to irradiation. The survival of GSCs play an important role on recurrence of glioma. The phenomena of low-dose hyper-radiosensitivity were seen in several tumor cells. A low-dose-rate ultra-microfraction (ld-UmRT) of radiotherapy was used to treat the GSCs by us. The daily fractions of 2 Gy were divided to 10 ultra-microfractions and every 0.2 Gy ultra-microfraction separated by 3 min time intervals. Then, the dose rate of 4~6 Gy/min was created to a low-dose-rate of 0.07 Gy/min. The survival rate decreased significant in GSCs after ld-UmRT compared to conventional radiotherapy in vitro. The most lethal among radiation-induced DNA lesions is the double-strand break (DSB). DSB repair is critical for cell survival after radiation induced DSB. There are data suggesting that EGFR-Akt-PKcs signaling pathways played an important role in radiosensitivity of tumor cell such as glioma cell and so on. We found that EGFRvⅢ was expressed in GSCs and the active of PKcs was inhibited in GSCs after GSCs received ld-UmRT. Therefore, We hypothesis that the DSB repair of GSCs depended on EGFRvⅢ/Akt/PKcs signaling pathways and this pathways was inhibited by ld-UmRT in GSCs. The EGFRvⅢ/Akt/PKcs signaling pathways will be tested in GSCs after ld-UmRT and the effect of ld-UmRT or conventional radiotherapy on blockage of EGFRvⅢ/Akt/PKcs signaling pathways in GSCs will be observed in our future work. The results of this study will help us to understand the mechanism of ld-UmRT increasing damage in GSCs.

胶质瘤干细胞（GSCs）放疗抵抗是胶质瘤复发和治疗失败的重要原因。基于肿瘤细胞低剂量放疗超敏现象，我们建立新的放疗方法、低剂量超微分割放疗(ld-UmRT)，即采用0.2Gy超微分割，每个超微分割间隔3分钟。前期研究结果显示ld-UmRT显著增强对GSCs杀伤作用。基于EGFR-Akt-PKcs信号通路在DNA双链断裂修复、细胞存活中的关键作用，结合我们前期发现GSCs表达EGFRvⅢ和ld-UmRT后GSCs内PKcs活化受抑的证据，推测EGFRvⅢ-Akt-PKcs途径在ld-UmRT克服GSCs放疗抵抗中起重要作用。拟通过体内外实验观察：GSCs在ld-UmRT后EGFRvⅢ-Akt-PKcs信号通路表达、活化；干预该信号通路后对GSCs在ld-UmRT及常规放疗后损伤效应变化。通过本研究，不但有利于完善GSCs放疗抵抗的分子机制，也将为ldUmRT特异性杀伤GSCs应用提供理论依据

EGFR高表达于约40%的人脑胶质母细胞瘤（Glioblastoma，GBM），其缺失外显子2-7的胞外段变异体EGFRvIII表达于20~30%的GBM患者。EGFRvIII具有不依赖配体结合即自身活化的重要特征，GBM细胞EGFRvIII表达是肿瘤细胞增殖、治疗抵抗的重要因素。.我们通过研究证实了EGFRvIII在人胶质瘤U251细胞的促增殖作用，并进一步发现EGFRvIII的促增殖作用是通过促进细胞分裂相关丝裂原蛋白CEP55的表达实现。抑制CEP55的表达可以逆转EGFRvIII在胶质瘤U251细胞的促增殖作用。该研究结果为针对表达EGFRvIII的胶质瘤细胞的靶向治疗选择提供了有力的支持，也为通过抑制CEP55的表达抑制胶质瘤细胞的增殖提供了靶向治疗新方向。.EGFR及EGFRvIII下游的转录因子Egr-1具有电离辐射启动其表达的特点。文献报道的研究结果提示Egr-1具有抑制肿瘤细胞增殖的作用，其表达增高与预后佳正相关。而我们通过检测胶质瘤患者及分析TCGA数据库，发现尽管在胶质瘤细胞Egr-1表达显著低于正常脑组织，但其在细胞接受电离辐射后表达显著增加，而Egr-1表达的增加显著地促进了胶质瘤U251细胞的增殖，通过持续的抑制U251细胞内Egr-1的表达，细胞的增殖显著下降，且Egr-1的促增殖作用是通过直接与增殖调控基因CCND1的启动子序列结合促进其表达实现。由此，通过我们的研究证实了胶质瘤细胞表达EGFRvIII后，细胞通过Egr-1、CCND1、CEP55等分子机制促进了细胞的增殖和放疗抵抗。为下一步逆转胶质瘤的放疗抵抗及抑制胶质瘤细胞的增殖临床研究提供了有力的理论基础。