阿司匹林乙酰化修饰ALDH1选择性清除大肠癌干细胞的分子机制研究

Clinical and epidemiological datas have revealed that Aspirin can significantly reduce the incidence and mortality of colorectal cancer(CRC),and improve the patients' prognosis. Many studies also suggest that CRC carcinigenesis and progression are closely associated with the existance of cancer stem cell (CSC). In addition,ALDH1 is one of the key CCSC markers involving tumor cell growth and differeciation. But it is still unknown whether Aspirin has effect on colorectal CSC (CCSC)as well as how it affects CCSC. Our group firstly found that Aspirin can eradicate CCSC selectively. We also found that Aspirin can restrain the enzyme activity of ALDH1 on a COX-2 independent manner, and the acetylation of ALDH1 can occur. Hence, we assume that Aspirin supresses the enzyme activity of ALDH1 through the acetylation of ALDH1,and further kills CCSC. Based on our previous results, we firstly screen and identify the mode and site of ALDH1 acetylation mediated by Aspirin ,which consequntly results in the inactivate of ALDH1 by immunoprecipitation and acetylation reporter system; Secondly, we analyze the biological significance of this modification; and then clarify the HDAC deacetylating the ALDH1 lysine residues and illuminate the target proteins interacting with acetylated ALDH1;finally verify the scientific design that Aspirin exerts its selective inhibition on CCSC through acetylated ALDH1. This study will afford a new target in CCSC interference therapy and establish a novel mechanism of Aspirin anti-CRC effect.

临床上Aspirin具有明确的防治多种肿瘤尤其是结直肠癌（CRC）的作用，已证实CRC发生发展与肿瘤干细胞(CSC)密切相关，其中ALDH1酶是结直肠癌CSC（CCSC）特征性标志物之一，也是其生长、分化的必需分子，因此探讨Aspirin如何抑制ALDH1酶活性清除CCSC也是CRC防治研究的重点。我们前期研究首次发现Aspirin能选择性清除CCSC，可通过非COX2依赖途径抑制ALDH1活性，同时ALDH1发生乙酰化，但机制不清也无报道。为此，我们假设含乙酰基团的Aspirin通过乙酰化ALDH1来抑制活性进而选择清除CCSC。本课题拟在前期研究的基础上，通过免疫沉淀及乙酰化报告系统等技术筛选鉴定Aspirin介导ALDH1活性降低的乙酰化方式、位点及生物学意义；明确与乙酰化ALDH1相互作用相关HDAC及靶蛋白，为Aspirin靶向清除CCSC提供新靶点,为解析抗CRC效应提供新机制

研究目的：临床上阿司匹林具有明确的防治多种肿瘤尤其是大肠癌的作用，已证实大肠癌发生发展与大肠癌干细胞密切相关。因此，阿司匹林可能通过抑制了大肠癌干细胞来减少大肠癌发生，提高患者生存率。..研究方法：利用免疫荧光和流式细胞分析技术在细胞模型和患者样本中分析阿司匹林治疗后大肠癌干细胞的比例变化。通过化学合成生物素化的阿司匹林筛选和鉴定与阿司匹林相互作用的靶蛋白；并利用CRISPR技术和光遗传学方法基因特异位点乙酰化修饰系统研究阿司匹林选择性清除大肠癌干细胞的作用机制；并通过小鼠异种移植模型体内证明阿司匹林和奥沙利铂在清除大肠癌干细胞的联合作用。..研究结果：我们首次证实阿司匹林除了降低大肠癌干细胞的数量，还可抑制大肠癌干细胞特性如克隆球形成能力及体内成瘤能力，增加其对奥沙利铂的敏感性，提示阿司匹林对大肠癌干细胞有较强的抑制作用。进一步发现我们阿司匹林抑制了 ALDH1阳性的 大肠癌干细胞，可以通过非 COX2 依赖的途径抑制 ALDH1 的酶活性。阿司匹林主要通过诱导大肠癌干细胞凋亡，抑制肿瘤进展，并增强化疗药物的抗肿瘤作用。此外，阿司匹林直接与细胞核中的p300相互作用，促进H3K9乙酰化，激活FasL表达，诱导大肠癌干细胞的细胞凋亡。值得注意的是，阿司匹林在普通大肠癌中不存在的这些效应的原因是普通的大肠癌细胞中H3K9被高甲基化的。此外，阿司匹林可以抑制奥沙利铂富集的CSCs，并可作为大肠癌的辅助治疗手段。..结论：我们揭示了阿司匹林消除大肠癌干细胞的一种独特的表观遗传学分子机制（p300-AcH3K9-FasL），该机制不依赖于cox的途径。这些发现更新了阿司匹林清除大肠癌干细胞的机制。