食管癌是全球最常见的消化道恶性肿瘤之一，据国家癌症中心的数据显示，中国食管癌新发病例和死亡病例每年占全球总病例数的50%以上，食管鳞状细胞癌（简称“食管鳞癌”）是中国食管癌患者的主要病理 类型，占所有食管癌患者的90%以上。食管鳞癌缺乏特异性诊断生物标 志物和有效治疗靶点，导致其早期诊断困难和治疗手段有限，这构成了我国食管鳞癌诊断和治疗领域的主要难题。基因组测序技术的应用推动了肿瘤精准治疗的发展，大量研究表明食管鳞癌的主要驱动因素为抑癌基因的失活，但其遗传驱动因素仍难以作为治疗靶点。因此有必要从新的角度探索和发展治疗线索。本研究基于基因组学和表观遗传组学等多组学测序结果，从遗传和表观遗传两个角度深入解析食管鳞癌发生发展的分子机制，旨在为食管鳞癌的个性化精准治疗提供新的理论基础。

    本论文第一部分对663例食管鳞癌及其配对样本的全基因组测序数据进行分析，结果发现NFE2L2基因是食管鳞癌中发生显著突变的基因，突变频率为6.5%，重要的是，NFE2L2基因突变是食管鳞癌患者不良预后的独立危险因素。体外表型实验表明部分NFE2L2突变类型会促进食管鳞癌细胞的迁移侵袭能力；进一步结合全转录组数据分析，发现NFE2L2突变通过促进细胞周期运转、抑制促炎反应、增强谷胱甘肽代谢等信号通路促进食管鳞癌的发生和发展。

    肿瘤的发生发展是一个复杂的过程，不仅涉及到遗传变异，表观遗传修饰也起到了至关重要的作用，调控基因活性的不同表观遗传学机制研究已成为生物学和肿瘤学研究的首要层级，增强子/超级增强子作为基因转录和表观遗传调控领域的关键组成部分，在肿瘤的发生发展中也发挥了不可忽视的作用。本论文第二部分研究通过对正常食管上皮细胞系和食管鳞癌细胞系进行H3K4me1和H3K27ac的ChIP-seq，应用计算框架绘制出食管鳞癌特异性超级增强子图谱，进一步结合多组学数据鉴定超级增强子驱动的致癌基因，结合体内体外实验我们确定了具有致癌作用的五个基因：KCNK1、PAPL、ENKUR、HAS3和CREG2，为进一步解析超级增强子激活致癌基因的分子机制，我们重点关注于驱动超级增强子调控的关键转录因子研究。KLF5作为驱动食管癌超级增强子调控的关键转录因子，我们发现其可以结合在KCNK1和PAPL的超级增强子区域以增强基因转录表达，从而促进肿瘤细胞的增殖，同时我们基于此调控通路发展了表观遗传药物联合治疗策略，使用靶向超级增强子区域的泛抑制剂JQ1和KCNK1的小分子抑制剂Quinidine可以显著抑制食管鳞癌PDX的生长。除验证KLF5的功能外，我们还基于信号网络分析和motif富集分析，新鉴定到HOXC13这一影响食管鳞癌转移侵袭的关键转录因子，我们发现HOXC13在食管鳞癌组织和食管鳞癌细胞系中均呈高水平表达；为进一步明确其致癌功能，我们构建稳定敲降HOXC13的食管鳞癌细胞系，开展体外细胞迁移侵袭实验和体内肺转移小鼠模型实验，初步证实了HOXC13高表达可以增强食管鳞癌细胞的迁移侵袭能力。HOXC13的ChIP-qPCR实验证实了HOXC13结合在ENKUR和HAS3靶基因的超级增强子区域，RT-qPCR实验亦证实了HOXC13对ENKUR和HAS3基因表达的调控作用，同时体外功能实验也表明稳定敲降ENKUR和HAS3表达可以减弱食管鳞癌细胞的迁移侵袭能力。以上研究较为全面地揭示了食管鳞癌特异性超级增强子促进肿瘤发生发展的调控网络。

    综上所述，本课题从遗传和表观遗传两个角度深入解析了食管鳞癌发生发展的分子机制，一方面研究发现NFE2L2基因突变是食管鳞癌患者不良预后的独立危险因素，另一方面我们从非突变表观遗传重编程角度鉴定并解析了食管鳞癌中超级增强子的作用机制，为食管鳞癌患者的个体化精准治疗提供了理论基础。

【关键词】食管鳞癌；遗传和表观遗传；NFE2L2突变；超级增强子；表观遗传治疗

Esophageal cancer is one of the most prevalent malignancies of the digestive system globally. Data from the National Cancer Center indicated that China accounts for more than 50% of the annual new cases and deaths from esophageal cancer worldwide. In China esophageal squamous cell carcinoma is the primary pathological type of esophageal cancer, accounting for over 90% of cases. ESCC is highly invasive, has a poor prognosis, lacks specific biomarkers and effective targets, making early diagnosis difficult and treatment options limited, which are major bottlenecks in the diagnosis and treatment of ESCC in China. The application of genome sequencing technology has promoted the development of tumor precision therapy, and numerous studies indicated that the main drivers of ESCC are the inactivation of tumor suppressor genes. However, the genetic drivers of ESCC remain non-targetable, thus necessitating the identification and development of new therapeutic clues from different dimensions. This study based on multi-omics sequencing results, delves into the molecular mechanisms of ESCC development from genetic and epigenetic perspectives, seeking to establish a novel theoretical foundation for personalized and precise treatments of ESCC.

The first part of this thesis analyzed whole-genome sequencing data from 663 cases of ESCC and their paired samples, identifying the NFE2L2 gene as a significantly mutated gene with a mutation frequency of 6.5%. Importantly, NFE2L2 mutations were identified as an independent risk factor for adverse prognosis among ESCC patients. In vitro phenotypic experiments showed that some NFE2L2 mutations types enhanced the invasive and migratory abilities of ESCC cells. Further analysis of whole-transcriptome data revealed that NFE2L2 mutations promoted the development of ESCC by enhancing cell cycle progression, inhibiting pro-inflammatory responses, and boosting glutathione metabolism.

Tumor development is a complex process involving not only genetic mutations but also crucial epigenetic modifications. Studies on various epigenetic mechanisms regulating gene activity have become a primary focus in biology and oncology. Enhancers/super-enhancers, as key components of gene transcription and epigenetic regulation, play an essential role in tumor development. The second part of this thesis studied the specific super-enhancer landscape of ESCC by conducting ChIP-seq of H3K4me1 and H3K27ac in normal esophageal epithelial cell lines and ESCC cell lines. By integrating multi-omics data, super-enhancer-driven oncogenes were identified, and using in vitro and in vivo experiments, five carcinogenic genes: KCNK1, PAPL, ENKUR, HAS3, and CREG2 were confirmed. Focusing on the key transcription factors driving super-enhancer regulation, KLF5 was found to bind to the super-enhancer regions of KCNK1 and PAPL to enhance gene transcription, promoting tumor cell proliferation. A combined epigenetic drug treatment strategy was developed using the pan-inhibitor JQ1 targeting super-enhancer regions and the small molecule inhibitor Quinidine of KCNK1, which could significantly inhibit the growth of ESCC PDX. Besides validating the function of KLF5, new key transcription factors influencing ESCC metastasis and invasion, such as HOXC13, were identified. High expression of HOXC13 in ESCC cells and tissues, compared to normal tissues or cells, promoted the invasive and migratory capabilities of ESCC cells. ChIP-qPCR experiments confirmed that HOXC13’s binding at the super-enhancer regions of the target genes ENKUR and HAS3, and RT-qPCR experiments confirmed its regulatory effects on ENKUR and HAS3. Functional experiments showed that stable knockdown of ENKUR and HAS3 expression could inhibit the invasive and migratory abilities of ESCC cells. The above study comprehensively revealed the regulatory network by which esophageal squamous cell carcinoma-specific super-enhancers promoted tumor initiation and progression.

In summary, this project deeply analyzed the molecular mechanisms of ESCC development from genetic and epigenetic perspectives. On one hand, it found that NFE2L2 gene mutations independently predict a worse prognosis for patients with ESCC. On the other hand, it identified and elucidated the molecular mechanism of super-enhancers in ESCC, providing a theoretical basis for the personalized precision treatment of ESCC patients.

Keywords：Esophageal squamous cell carcinoma; Genetics and epigenetics; NFE2L2 mutation; Super-enhancers; Epigenetic therapy