肝癌干细胞LSD1蛋白逃逸E3泛素连接酶FBW7降解导致BET抑制剂耐药的机制研究

Inhibitors of BET (bromodomain and extra-terminal) family proteins are newly designed epigenetically targeted drugs that selectively abrogate the binding of BET proteins to acetylated histones, thus down-regulating transcription of critical oncogenes, such as MYC. Although these BET inhibitors show promise in treating a series of human hematologic and solid cancers, drug resistance is emerging thus it is critical to dissect the underlying mechanisms. We have identified liver CICs (cancer-initiating cells) as a key subset of cancer cells insensitive to BET inhibitor and that highly expressed levels of LSD1 (lysine-specific histone demethylases 1) in CICs maintain their resistance phenotype, whereas the molecular basis of LSD1 high expression in CICs remains unknown. In light of the preliminary data showing that LSD1 protein might have escaped the ubiquitination and proteasomal degradation machinery facilitated by the E3 ligase FBW7, we propose that highly activated Wnt/β-catenin pathway may inhibit the kinase activity of GSK3-α/β to abrogate FBW7 mediated-ubiquitination/proteasomal degradation of LSD1, thus contributing to the refractoriness of CICs to BET inhibitors. To test this hypothesis, a series of experimental techniques including CRISPR-cas9 gene knockout and site mutagenesis will be applied to examine the molecular mechanisms of GSK3-α/β-depedent ubiquitination of LSD1 by FBW7. Moreover, we will evaluate whether evasion of this protein degradation signaling axis by liver CICs is causative to the failure of BET inhibitor treatment. Together, this research proposal might provide novel cellular and molecular targets for optimizing caner precision therapy using BET inhibitors.

BET蛋白BRD结构域抑制剂是新兴的表观靶向药物，抑癌效果显著但耐药现象突显，亟需解析耐药机制以提高疗效。以肝癌为模型，我们发现肿瘤干细胞是BET抑制剂耐药的关键细胞亚群，其组蛋白去甲基化酶LSD1高表达是耐药的重要原因，但肝癌干细胞LSD1表达上调的分子机制不详。由于LSD1蛋白可能逃逸E3泛素连接酶FBW7介导的泛素-蛋白酶体降解，我们提出假说：肝癌干细胞Wnt/β-catenin信号抑制GSK3-α/β激酶活性，使FBW7无法促进LSD1蛋白泛素化降解，导致BET抑制剂耐药。本项目拟利用CRISPR基因敲除、定点突变等技术和动物实验，探讨FBW7依赖GSK3-α/β降解LSD1蛋白的机制，以及肝癌干细胞逃逸此机制导致BET抑制剂耐药的效应，为基于BET抑制剂的肿瘤精准治疗，提供新的细胞靶点和分子靶标。

BET蛋白BRD结构域抑制剂是新兴的表观靶向药物，抑癌效果显著但耐药现象突显，亟需解析耐药机制以提高疗效。本项目深入研究蛋白翻译后修饰，尤其是泛素化修饰调控BET抑制剂耐药特性和肿瘤恶性生物学行为的细胞和分子机制，重点关注重要的表观调控因子如组蛋白去甲基化酶LSD1高表达和表观识别蛋白BRD4的作用机理。.我们研究发现组蛋白去甲基化酶LSD1蛋白可以与E3泛素连接酶FBW7结合，因此提出假说：肝癌干细胞Wnt/β-catenin信号抑制GSK3-α/β激酶活性，使FBW7无法促进LSD1蛋白泛素化降解，导致BET抑制剂耐药。然而在验证过程中，我们发现FBW7虽能结合LSD1蛋白，但不直接导致其降解，提示LSD1蛋白不是FBW7真正的底物蛋白。鉴于此种情况，我们迅速调整研究策略，建立了高通量的CRISPR筛选系统，发现新的LSD1泛素化修饰酶ZER1，深入研究ZER1降解LSD1的机制，取得创新学术发现。在本项目的支持下，我们还深入探讨了肿瘤中BET抑制剂的重要靶分子—BRD结构域蛋白影响耐药关键蛋白Snail泛素化修饰的分子机制。我们发现，BRD4肿瘤组织高表达与肿瘤进展和耐药密切相关，可通过识别Snail上的K146及K179乙酰化修饰，驱逐E3泛素连接酶β-TRCP和FBXL14与Snail的结合，使Snail免于泛素化和蛋白酶体降解的作用。经典BET抑制剂JQ1可大幅降低BRD4的上述效应，因此这可能是导致BET抑制剂耐药后肿瘤快速进展的重要机制。.本项目相关研究成果发表在Cell Death and Differentiation (IF: 10.717)、Cancer Research (IF: 9.727)、Cancer Letters (IF: 7.360)、Gastric Cancer (IF: 7.088)等国际肿瘤研究经典期刊（申请人均为通讯作者，文章均已标注项目基金号NSFC 81672463）。研究成果在肿瘤治疗领域，特别是在肿瘤靶向药物开发领域具有一定的转化应用价值，为基于BET抑制剂的肿瘤精准治疗，提供了新的细胞靶点和分子靶标。