APE1结合G4/8-oxoG位点调控EMT在肺癌靶向治疗耐药中的机制研究

Target therapy of lung cancer, including EGFR-TKI treatment, eventually develops acquired resistance after average 8-12 months, which restricts the clinical benefit of these population of lung cancer patients. Our previous studies have shown that apurinic/apyrimidinic endonuclease (APE1) is an important DNA repair molecule closely correlated with epithelial-mesenchymal transition mediated acquired EGFR-TKI resistance, but the mechanism of how APE1 regulates EMT-related genes expression simultaneously remains unclear. Cancer cells produce a number of oxidizing base damage (8-oxo-G) during EGFR-TKI treatment, recent study has identified that 8-oxo-G formed in the promoter region G-quadruplex(G4) sequence may be a novel epigenetic marker, but its biological significance and the detailed mechanism is largely unknown. Based on our previous research and preliminary experimental results, we put forward a scientific hypothesis: APE1 "bounding without incision" at the 8-oxo-G sites in the promoter region, which promotes the epigenetic regulation of EMT-related genes and in turn facilitates the EGFR-TKI resistance. This study intends to mapping the distribution features of 8-oxo-G sites with transcription activity after EGFR-TKI treatment, through APE1/G4-ChIP-seq, APE1-sensitive sequencing and RNA-seq cross comparison technology, and focuses on the molecular mechanisms of APE1 in 8-oxo-G associated epigenetic regulation sites forming in the G4 sequence located in promoter region. The project will elucidate the biological significance of epigenetic markers as 8-oxo-G in EGFR-TKI resistance of lung cancer, and reveal the novel molecular mechanisms of APE1 in the gene transcription regulation, which further provides experimental basis for APE1-based EGFR-TKI resistance overcoming in lung cancer.

肺癌靶向治疗存在不可避免的获得性耐药，制约其临床获益。我们前期研究显示脱嘌呤脱嘧啶核酸内切酶（APE1）通过调控上皮间质转化（EMT）参与了EGFR-TKI耐药，但分子机制仍不清楚。新近发现启动子区G四联体（G4）序列中形成的氧化性碱基损伤（8-oxo-G），是一种全新的表观遗传学标记，但其生物学意义尚不明确。我们首次提出，APE1对G4序列中8-oxo-G位点“结合而不切除”促进EMT相关基因表观遗传学调控而介导了肺癌EGFR-TKI耐药。本项目拟通过APE1/G4-ChIP-seq和APE1酶切测序交叉比对阐明8-oxo-G在基因组中分布特征，重点研究APE1在G4序列8-oxo-G形成表观遗传学调控位点中的分子机制。该项目有助于阐明8-oxo-G位点在EMT相关基因表达调控中的生物学意义，揭示APE1参与基因转录调控中的新机制，为以APE1为靶点逆转靶向治疗耐药提供理论依据。

对于携带驱动基因突变的肺癌患者，靶向治疗具有很高的客观缓解率，也极大的延长了患者的无疾病进展生存期。然而靶向治疗也不可避免出现获得性耐药，而其中肿瘤细胞发生上 皮 -间 充 质 细 胞 转 化（Epithelial-mesnchymal transition, EMT）和肿瘤干细胞(Cancer Stem Cells, CSCs)是癌症耐药重要的分子机制。尽管越来越多的转录调控机制也被证实参与了EMT和CSCs的发生过程，然而仍然无法完全解释这过程中基因表达的剧变机制。.APE1是BER通路的关键限速酶，是具有核酸内切酶和氧化还原双功能蛋白酶，OGG1同样是双功能酶。AP和8-oxo-G位点分别为APE1和OGG1 酶底物。BER蛋白已被证明具有超越 DNA 修复的作用，在表观遗传中有新的发现。我们运用APE1抗体和OGG1抗体抓取到APE1和OGG1特异性结合在基因组中的AP位点和8-oxoG位点邻近的DNA片段并进行高通量测序，绘制全基因组DNA碱基损伤（8-oxo-G/AP 位点）热点图谱，结果发现APE1和OGG1蛋白结合区域呈现显著不均匀性并且趋向基因启动子区域分布，APE1和OGG1结合的motif高度匹配癌症相关基因转录因子，APE1和OGG1蛋白富集区域的基因与癌症密切相关，还发现APE1和OGG1与组蛋白结合区域部分重叠。本研究成功用APE1和OGG1抗体结合ChIP-seq的方法，绘制相应全基因组富集区域图谱，表明碱基损伤位点并非随机分布于基因组中而是与启动子区密切相关，因此推测 8-oxo-G/AP 位点可能是潜在的表观遗传标志物。通过HCC827和HCC827ER 细胞全转录组测序(RNA-sequencing，RNA-Seq)分析，结合基因富集分析（Gene Set Enrichment Analysis，GSEA），将所有表达基因和预设的EMT和Stem-like相关的基因集比对分析，结果表明HCC827ER相对 HCC827的差异基因在EMT和Stem-like基因集的富集分数均在0.5左右，提示EGFR-TKI耐药与EMT和干细胞相关基因密切相关。.综上，APE1 和 OGG1 作为新型表观遗传标志物 8-oxo-G/AP 位点结合蛋白可能通过 全新的表观遗传调控机制促使EGFR-TKI耐药。