胃癌细胞LSD1与EMT通路相关蛋白相互作用的机制及小分子抑制剂干预研究

Histone lysine specific demethylase 1 （LSD1） can selectively demethylate mono, di-methylated histone H3K4 and H3K9 and regulate the expression and activity of the downstream proteins through altering the chromatin structure. LSD1 has been considered a potential anti-tumor therapeutic target. It was reported to be overexpressed in several kinds of solid tumors, including gastric caners. Meanwhile, down regulation of LSD1 expression with RNAi or inhibiting the activity of LSD1 by inactivators can prevent the growth, migration and invasion of cancer. In our previous studies, we found that the expression of TGF β1 (Transforming growth factor beta 1) and Wnt are positively correlated with LSD1 expression in human gastric tumors. Hence, LSD1 inhibitors could be applied to block the interaction between LSD1 and TGF β1 or Wnt, inactivate TGF β1- or Wnt-induced EMT (Epithelial-Mesenchymal Transition) and activate aberrant silenced cancer suppresors. In the current project, the role of the interaction between LSD1 and TGF β1 or Wnt, their mutual regulation in cancer development and progression will be investigated in cellular level, nude mouse and LSD1 deficient mouse. With computer aided drug design method, potent and selective LSD1 inhibitor will be designed and synthesized in order to modulate the protein-protein interaction with LSD1 as a key factor, as well as to study the mechanism of LSD1 inhibitor’s action during EMT. Our major goal is to further demonstrate the mechanism of EMT and acquire more potent and effective LSD1 inhibitors for cancer therapy.

组蛋白赖氨酸特异性去甲基化酶1(LSD1)可特异性去除组蛋白赖氨酸H3K4、H3K9单双甲基，通过改变染色质结构，调节其下游蛋白表达, 为目前抗肿瘤研究前沿和热点。我们前期研究证实LSD1在胃癌等多种肿瘤中高表达，LSD1特异性抑制剂或RNAi 介导表达下调可抑制肿瘤细胞生长, 侵袭和转移。本课题通过细胞及整体动物水平研究探讨1.胃癌发生发展过程中LSD1与TGF β1/SMADs通路、ERK/NF-κB/TWIST通路、Wnt/β-Catenin通路中相关关键蛋白相互作用，揭示LSD1调控EMT过程的作用机制；2.通过计算机辅助药物分子设计合成及活性筛选获得高效高选择性LSD1小分子抑制剂，干扰上述EMT相关蛋白蛋白相互作用调控通路，为揭示实体肿瘤恶性增殖及EMT过程相关蛋白相互调控作用的分子机制提供重要理论依据，并发现新的调控EMT过程蛋白蛋白相互作用机制和高生物活性小分子抑制剂。

LSD1作为表观遗传领域关键蛋白，可选择性去除H3K4和H3K9的甲基，通过改变染色质结构调节下游靶基因表达，发挥其癌基因的作用。本项目在前期研究基础上，首次阐述了LSD1与TGF β1相互调节促进胃癌转移的分子机制，并从性别差异方面首次提出LSD1对胃癌转移的差异调节作用机制。同时，本项目发现了外泌体LSD1对靶细胞干性的促进作用及其对化疗敏感的抑制作用，首次提出外泌体LSD1可作为预测临床化疗敏感性及响应率的标志物。在此基础上，本项目利用基于结构的药物设计，合成、筛选获得多个高效小分子LSD1抑制剂，通过抑制LSD1活性抑制胃癌转移。本项目相关研究发现了胃癌转移及耐药的新机制，证实LSD1抑制剂可能做为胃癌临床靶向治疗的新策略，为预测胃癌化疗敏感性、响应率及患者预后提供了新方法。.本项目主要工作及成果包括以下4个方面：.1）建立了包含1000对临床样本的样本库，发现低分化胃癌患者瘤组织中LSD1与TGF β1表达量正相关，确证了TGF β1通过激活ERK1/2-NF-κB-p300通路激活LSD1启动子上调LSD1表达；而LSD1基因敲除可通过抑制TGF β1启动子附近H3K9me1/2的丰度抑制TGF β1，从而抑制胃癌细胞EMT过程，抑制胃癌细胞转移。.2）发现在LSD1高表达的男性胃癌组织中Wnt3a与EMT相关蛋白Vimentin、Snail、N-Cadherin高度相关，而女性胃癌患者中上述蛋白相关性不显著；进一步作用机制研究发现男性胃癌细胞MGC-803中LSD1促进Wnt3a分泌，LSD1 KO则可通过抑制Wnt3a分泌相关蛋白PORCN表达从而抑制Wnt3a分泌，进而抑制β-catenin及Snail入核及EMT相关蛋白表达，抑制胃癌转移。.3）首次发现核蛋白LSD1以外泌体蛋白的形式被分泌到细胞外，以外泌体为载体进入到靶细胞中，促进靶细胞干性相关蛋白表达，抑制靶细胞化疗敏感性。.4）通过计算机辅助药物设计及构效关系分析，合成了400个嘧啶及嘧啶并三氮唑类衍生物。通过活性筛选，蛋白及细胞水平机制研究，发现了一系列能够高效抑制LSD1活性的先导化合物，其中活性最好的化合物II-15u的IC50为50nM。该化合物不仅能够抑制LSD1高表达胃癌细胞的增殖，同时可以诱导白血病细胞分化，发挥抗肿瘤作用。