程序性死亡配体PD-L1调控宫颈癌放疗敏感性机理研究

Radio-resistance is one of the main causes of uterine cervical cancer recurrence, so unveiling the underlying mechanism of radio-resistance and exploring new therapeutic targets is the key to effectively prevent and treat cervical cancer recurrence. Previous studies in this project found that programmed death ligand 1 was significantly up-regulated in tissues of cervical cancer patients with radio-resistance and this up-regulation was negatively correlated with prognosis. This indicates PD-L1 plays a regulatory role in the process of radio-resistance in cervical cancer. Vitro experiments revealed that PD-L1 may affect the radio-sensitivity of cervical cancer cells by regulating the glycolysis process. Subsequent studies revealed that PD-L1 may activate downstream FAK/AKT1/GSK3β signaling pathway through ITGB4 to regulate the phosphorylation level of hexokinase 2, thereby regulating the level of glycolysis and radio-sensitivity of cervical cancer cells. However, the mechanism is still unclear. Based on the previous work, this project intends to study the regulation mechanism of PD-L1 and its downstream pathways on the sensitivity of cervical cancer radiotherapy by vitro and vivo experiments such as gene cloning, RNA-seq, protein expression and purification, glucose metabolism associated experiments based on works we had done before. This project is of important theoretical significance and potential clinical conversion value for clarifying the mechanism of cervical cancer radio-resistance and exploring novel molecular targets that can be used for radio-sensitivity evaluation and treatment for cervical cancer patients.

放疗抵抗是宫颈癌复发的主要原因之一, 故揭示放疗抵抗机制并探寻新的治疗靶点是有效预防和治疗宫颈癌复发的关键。本项目前期研究发现程序性死亡配体1在产生放疗抵抗的宫颈癌患者组织中表达水平显著上调，与患者预后呈负相关，这表明PD-L1可能在宫颈癌放疗抵抗产生过程中发挥一定的调控作用。体外实验揭示PD-L1可能通过调控糖酵解过程来影响宫颈癌细胞放疗敏感性。随后研究发现PD-L1可能通过ITGB4激活下游FAK/AKT1/GSK3β信号通路来调节己糖激酶2磷酸化水平，进而调控糖酵解水平和宫颈癌细胞的放疗敏感性，但具体调控机制仍不明确。据此，本项目拟在前期工作基础上利用分子克隆、基因芯片、蛋白表达及纯化、糖代谢等体内外实验来研究PD-L1及下游信号通路对宫颈癌放疗敏感性的调控机制。这对于明确宫颈癌放疗抵抗和寻找可用于放疗敏感性评估和治疗的分子靶点具有重要的理论意义和潜在临床转化价值。

宫颈癌是女性生殖系统常见恶性肿瘤，放疗在宫颈癌的治疗当中具有重要的临床价值。对于早期宫颈癌患者，可先行手术，术后根据病检结果，部分患者需追加放疗，而对于中晚期患者，可直行放射治疗。因此，在临床实践中，放疗具有重要的意义。随着临床实践的进行，宫颈癌的放射抗拒问题逐渐凸显，成为患者疾病复发、预后欠佳的重要原因。因此探讨宫颈癌的放射抗拒机理是本研究的重点。PD-L1在肿瘤细胞中具有重要的意义，为肿瘤免疫治疗开启新篇章。在申请人的前期研究中，我们已证实宫颈癌PD-L1表达上调可影响肿瘤细胞的功能，促进肿瘤细胞的恶性代谢及转移。放疗通过射线导致肿瘤细胞坏死，能促进免疫细胞的浸润，具有一定程度的免疫微环境激活作用。肿瘤细胞PD-L1上调是否会引起宫颈癌的放疗敏感性的改变成为本研究关注的重点。本项目通过构建PD-L1差异表达细胞系，发现PD-L1可能通过有氧糖酵解过程参与宫颈癌放射抗拒调控。PD-L1表达上调，肿瘤放射治疗后凋亡减少，糖酵解水平提高。在此表型基础上，进一步研究发现PD-L1上调可活化下游GSK3β信号通路，β-catenin表达上调并出现入核现象，通过ChIP以及荧光素酶双报告基因等核心实验，我们确定β-catenin可作为转录因子结合HK2启动子，上调HK2表达，进一步增强肿瘤细胞无氧糖酵解，GSK3β信号通路活化最终影响能量代谢，促进宫颈癌放射抗拒的出现。本课题体内实验显示，PD-L1表达上调可促进宫颈癌细胞生长，而PD-L1相关ICI使用可显著抑制肿瘤的生长，起到一定的放疗增敏的作用。本课题揭示了宫颈癌放射治疗过程PD-L1参与的内在调控网络及其促进肿瘤放射抗拒的作用机制，并明确其中关键分子与临床病理及预后的相关性，这对于更好地明确放射抗拒机制、完善宫颈癌放射抗拒风险评估模型和寻找放射治疗增敏靶点具有重要的理论意义和潜在临床转化价值。