脂肪酸合成酶FASN调控TEAD4棕榈酰化诱导奥希替尼获得性耐药及蟾毒灵对其作用的机制研究

奥希替尼对EGFR突变肺癌患者有显著疗效，但耐药无法避免，耐药机制未被完全阐明，临床无有效防治措施。前沿研究显示，代谢紊乱及蛋白质棕榈酰化异常与肿瘤耐药密切相关。前期研究中我们应用RNA-seq、棕榈酰蛋白纯化技术/质谱分析发现：奥希替尼耐药细胞中脂肪酸合成酶FASN高表达，转录因子TEAD4棕榈酰化水平升高，YAP、ERBB3等下游通路活化；蟾毒灵下调FASN，减少TEAD4棕榈酰化，增强奥希替尼抗肿瘤效果。故推测：FASN介导TEAD4棕榈酰化，激活YAP、ERBB3等下游通路诱导奥希替尼耐药；蟾毒灵下调FASN，抑制TEAD4/YAP、ERBB3等通路，逆转奥希替尼耐药。本项目拟应用Acyl-RAC、活细胞成像、DNA点突变、荧光素酶报告等技术，从临床、动物、细胞层面探讨FASN介导TEAD4棕榈酰化诱导奥希替尼耐药机制及蟾毒灵干预作用，为开发抗癌中药及防治靶向药物耐药提供理论依据。

Osimertinib has marked efficacy in patients with EGFR-mutated lung cancer. However, the development of acquired resistance to osimertinib cannot be avoided. The precise mechanisms of resistance to osimertinib remain largely unknown. There’re not effective strategies to prevent and/or overcome acquired resistance to osimertinib. Metabolic pathway reprogramming and deregulated protein palmitoylation have been shown to support cancer cell proliferation,survival and therapeutic resistance. We established osimertinib-resistant cell lines by exposing sensitive cell lines to osimertinib. RNA-seq, ABE/MS and Western blots were performed to analyze global gene expression changes and palmitoylated proteins. We found the expression of fatty acid synthase (FASN) and the level of TEAD4 palmitoylation were sharply increased in osimertinib-resistant cell lines. KEGG pathway and Gene Set Pathway Enrichment analyses (GSEA) showed fatty acid metabolic pathway, YAP and ERBB3 signaling pathways were active in resistant cells. We also found that bufalin could down-regulate FASN and TEAD4 palmitoylation level in osimertinb-resistant cells. Based on these results, we therefore hypothesized that FASN induces the resistance to osimertinib through TEAD4 palmitoylation, activating its downstream YAP/ERBB3 signaling pathway; bufalin reverses osimertinib resistance by down-regulating FASN and inhibiting TEAD4-YAP/ERBB3 axis. We will reveal the mechanisms of FASN-induced resistance to osimertinib and bufalin reverses osimertinib resistance using Acyl-RAC, the Incucyte ZOOM live-cell analysis system, Co-IP, Deletion Mapping and Luciferase reporter assay. Our study will provide a novel strategy for development of new antitumor drugs and reversion of targeted drug resistance.