XPF基因遗传变异对非小细胞肺癌铂类化疗毒性的影响及其功能研究

Platinum-based regimes are the standard first-line chemotherapy for patients with advanced non-small cell lung cancer (NSCLC). However, the efficacy of platinum-based chemotherapy (PBC) varies remarkably among the patients with similar clinical background, and treatment compliance was hampered by severe toxicities. Nucleotide excision repair (NER) pathway is closely related to DNA damage repair-induced by chemotherapy. Previous studies have indicated that single nucleotide polymorphisms (SNP) in this pathway were associated with the efficacy and toxicity of chemotherapy in NSCLC patients, but the specific molecular mechanism is still unclear. Through a candidate gene approach, we found that A allele of the SNP rs1799798 in ERCC4/XPF, a key endonuclease in the NER pathway, was associated with chemotherapy toxicity and worse survival outcome, as well as a lower XPF mRNA expression. Bioinformatics analysis revealed that this SNP might affect the binding of transcription factors and Pol II polymerase to the DNA fragment. In vitro studies indicated that knockout of XPF impaired NER repair function and increased the cellular sensitivity to cisplatin. It is urgent for us to further reveal the molecular mechanism that this polymorphism, through regulating XPF gene expression, affects the ability to repair DNA damage induced by platinum chemotherapy, and ultimately causes toxic reactions and survival differences through experiments on clinical specimens, cells and animal models. Based on these initial findings, this project will provide a theoretical basis for the mechanism underlying the association between common genetic variant and platinum chemotherapy related outcome, and contribute to the development of a new biomarker for individualized lung cancer chemotherapy.

晚期肺癌患者的临床一线方案是铂类联合化疗，然而该方案对相同临床背景患者的远期疗效和毒性差异很大。核苷酸剪切修复（NER）通路和化疗后DNA损伤修复关系密切，既往研究发现该通路单核苷酸多态性（SNP）与非小细胞肺癌患者化疗的疗效及毒性相关，但具体分子机制尚不明确。通过候选基因策略，我们既往发现NER通路中关键内切酶XPF基因SNP位点可显著增加非小细胞肺癌患者铂类化疗毒性反应并降低生存率，且可能参与下调XPF mRNA表达。前期生物信息分析发现该SNP可能影响转录因子及Pol II聚合酶的结合。在肺成纤维细胞中敲除XPF可减弱NER通路修复能力并增强对顺铂的敏感性。后续亟待通过临床标本、细胞和动物实验，进一步揭示该多态性位点通过调控XPF基因表达，影响铂类化疗所致DNA损伤的修复能力，最终引起毒性反应及生存差异的分子机制。本项目基于临床病例研究，为辅助化疗的生物标志物研究提供依据。

本项目的背景基于中国人群非小细胞肺癌(NSCLC)患者生存相关的遗传易感性基因及其变异的发现和机制研究。我们分别在核酸切除修复（NER）、人类白细胞抗原（HLA）和免疫激活通路中发现了影响肺癌进展的关键易感基因及多态性位点，并对其生物学机制展开了功能研究。在NER通路中，我们发现位于XFP基因内含子的rs1799798（G>A）的A等位基因可显著增加接受铂类化疗的晚期肺癌患者的胃肠毒性，并缩短总生存期。在肺癌细胞株中（A549和BEAS-2b），我们通过利用包含XPF/rs1799798-A和-G基因型序列的双荧光素酶报告实验，发现含有G等位基因的内含子片段的荧光素酶的强度显著高于A等位基因，提示该单核苷酸的改变可能会减弱增强子的转录调控能力；进一步通过EMSA实验，初步证实了G基因型与核蛋白的结合能力较强，而G->A基因型的改变，可能会下调XPF表达而降低患者生存。在免疫激活通路中，我们发现VAV2和PSMA4的基因多态性位点与非小细胞肺癌病人的总生存相关。通过进一步的生物信息学和分子生物学分析，发现VAV/rs12002767的C等位基因型可能通过上调靶基因表达，影响免疫细胞浸润水平，提高肺癌患者的死亡风险。人类白细胞抗原 (HLA) 区域具有高度多态性，通过驱动抗原呈递、补体级联和白细胞成熟对抗癌细胞。我们发现在HLA通路中位于TAP2内含子区域的遗传变异位点与非小细胞肺癌患者的生存显著相关，并通过基于ChIP-seq和Hi-C公共数据的分析，发现该位点可能远端调控HLA-DQA1启动子影响该基因表达。验证了功能性遗传生物标志物促进肺癌发展，并影响病人预后提供了潜在的生物学机制。本项目的科学意义在于发现了关键的遗传易感性生物标志物并初步分析其潜在分子机制，这些生物标志物的发现有助于进一步提高对肺癌患者生存预测和易感基因的转化研究。