背景和目的：作为消化道常见的恶性肿瘤，食管鳞癌具有早期症状不明显和恶性程度高的特点，严重威胁人类健康。食管鳞癌患者预后差，除了复杂的发病机制外，对放化疗抵抗是导致食管鳞癌患者存活率低的一个重要原因，但是目前关于食管鳞癌放化疗抵抗的机制尚不明确。本研究目的在于探究潜在功能基因在食管鳞癌中的表达和调控机制，为食管鳞癌寻找新的潜在治疗靶点和准确的生物标志物去预测和改善放化疗治疗效应。

方法：本研究用慢病毒表达载体和CRISPR/Cas9基因编辑技术在食管鳞癌细胞系中进行目的基因表达的增高和降低。通过RT-qPCR检测细胞中的RNA表达水平，利用Western blot和免疫组织化学染色检测蛋白质表达水平。运用细胞活力检测、细胞克隆形成、极限稀释法、单细胞凝胶电泳等实验方法验证食管鳞癌细胞对于化疗药物或放射处理的敏感性。利用染色质免疫共沉淀、双荧光素酶报告基因实验和电泳迁移率实验检测转录因子与DNA的结合。m⁵C点印记实验和m⁵C-RNA免疫共沉淀-qPCR检测细胞中的m⁵C水平。另外，免疫缺陷小鼠细胞移植瘤模型、4-NQO诱导小鼠食管癌模型和PDX模型用于检测基因表达对肿瘤增殖和发生发展的作用以及抑制剂与细胞毒性药物的作用效果。最后在一组接受术前新辅助放化疗的患者队列中通过免疫组织化学染色分析目的基因表达与放化疗敏感性的关系。

结果：我们发现了与正常组织相比，在食管鳞癌中TIGAR的表达显著升高，高表达TIGAR的患者生存时间短。与Tigar^+/+和Tigar^+/-小鼠相比，Tigar^-/-小鼠在4-NQO诱导后食管上肿瘤的数量和大小显著降低；Nsun2^-/-小鼠在4-NQO诱导后得到相似的结果。TIGAR高表达的细胞和组织p-AMPK和GLS的表达水平更高，而且细胞毒性化学治疗药物对TIGAR高表达的细胞抑制作用不明显。TIGAR高表达的食管鳞癌细胞移植瘤模型、4-NQO诱导食管癌模型和PDX模型中使用谷氨酰胺酶抑制剂结合细胞毒性化学治疗药物比单一化学治疗更加有效。除此之外，我们发现RNA甲基转移酶NSUN2过表达可以上调TIGAR的表达，NSUN2敲除可以下调TIGAR表达，机制上，NSUN2通过m⁵C修饰增加了TIGAR mRNA的稳定性，从而促进了TIGAR表达。NSUN2在食管鳞癌中高表达是由于转录因子STAT1对其启动子区rs10076470不同基因型的差异结合导致的。TIGAR高表达细胞对IR处理不敏感，并且TIGAR过表达可减弱NSUN2敲除细胞的放射敏感性，TIGAR敲降可减弱NSUN2过表达细胞的放射抵抗性。在新辅助放化疗抵抗的食管鳞癌活检样本中TIGAR和NSUN2表达更高，两者的表达呈现显著正相关。

结论：TIGAR在食管鳞癌中高表达并促进肿瘤的发生发展，谷氨酰胺通路是TIGAR高表达食管鳞癌的治疗靶点。STAT1介导NSUN2的eQTL调控作用使其高表达，NSUN2高表达通过m⁵C修饰促进TIGAR mRNA稳定性从而促进TIGAR高表达导致食管鳞癌放射治疗抵抗。因此，TIGAR高表达介导食管鳞癌放化疗抵抗，而且TIGAR高表达可能作为食管鳞癌放化疗敏感性的预测指标及潜在的个体化治疗标志物。

Background & Aims: Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor of digestive tract in the worldwide and there are no obvious symptoms at its early stage, which seriously threatens human health. Chemoradiotherapy (CRT) resistance is an important reason for poor prognosis of ESCC in addition to the complex pathogenesis despite advances made in diagnostic techniques and multimodal treatments, in which the underlying mechanism is largely unknown. This study aims to investigate potentially functional mechanism post-transcriptional modification and regulation of TIGAR. It is urgent to identify new therapeutic targets and accurate biomarkers to predict and improve the CRT effects in ESCC.

Methods: Lentivirus expression vector and CRISPR/cas9 system were applied to increase and decrease the expression of target gene in ESCC. The RNA expression level was detected by real-time fluorescence quantitative PCR (RT-qPCR) and the protein expression level was detected by western blot and immunohistochemical (IHC) staining. The sensitivity of ESCC cells to chemotherapeutic drugs or radiation treatment was verified by cell viability assay, cell colony forming ability, extreme limiting dilution assays (ELDA) and single cell gel electrophoresis. We examined the interaction between protein and DNA by chromatin immunoprecipitation (CHIP), dual-luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA). m⁵C dot blot assay and m⁵C-RNA immunoprecipitation-qPCR (m⁵C-RIP-qPCR) were used to detect the level of m⁵C in ESCC cells. In addition, ESCC cell xenografts, 4-NQO induced ESCC and patient-derived xenografts (PDXs) were used to detect the effect of target gene in ESCC proliferation and development as well as the therapeutic effect of glutaminase inhibitor and cytotoxic chemotherapeutic drugs. Finally, IHC staining was used to analyze the relationship between gene expression and chemoradiotherapy resistance in a patient cohort receiving neoadjuvant CRT.

Results: In this study, the results showed that ESCC had TIGAR levels significantly higher than adjacent normal tissues and patients with high TIGAR level survived significantly shorter time than patients with low TIGAR level in ESCC. TIGAR reprogramed glucose metabolism from glycolysis to the glutamine pathway through AMP-activated kinase. Tigar knockout mice had reduced ESCC tumor burden and growth rates. Treatment of TIGAR-overexpressing ESCC cell xenografts and patient-derived tumor xenografts in mice with combination of glutaminase inhibitor and chemotherapeutic agents achieved significant more efficacy than chemotherapy alone. In addition, we identified an ESCC-specific cis-eQTL formed by rs10076470 G to A variation in the promoter of 5-methylcytosine (m⁵C) methyltransferase (NSUN2) gene that interacted with STAT1 and upregulated NSUN2 expression. We revealed that NSUN2 enhanced TIGAR production through mRNA-m⁵C modification that confered ESCC radioresistance by enhancing the DNA repair capacity. Significant correlations between NSUN2/TIGAR levels in ESCC and CRT vulnerability were observed in the patient cohort.

Conclusions: TIGAR is an important player in ESCC proliferation and progression. Glutamine pathway is a therapeutic target for TIGAR-overexpressing human ESCC. NSUN2 upregulates TIGAR expression by enhancing TIGAR mRNA m⁵C-modification. TIGAR overexpression confers CRT resistance and might be a marker to predict CRT vulnerability in ESCC.